GLP-1 derivatives II

ABSTRACT

The present invention relates to a derivative of GLP-1(7-C), wherein C is 35 or 36 which derivative has just one lipophilic substituent which is attached to the C-terminal amino acid residue.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT/DK99/00086 filed Feb. 24, 1999which claims priority under 35 U.S.C. 119 of Danish application 0274/98filed Feb. 27, 1998 and of U.S. Provisional application 60/084,357 filedMay 5, 1998, the contents of which are fully incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to novel derivatives of humanglucagon-like peptide-1 (GLP-1) and fragments thereof and analogues ofsuch fragments which have a protracted profile of action and to methodsof making and using them. The invention furthermore relates to novelderivatives of exendin and the uses of such derivatives.

BACKGROUND OF THE INVENTION

Peptides are widely used in medical practice, and since they can beproduced by recombinant DNA technology it can be expected that theirimportance will increase also in the years to come. When native peptidesor analogues thereof are used in therapy it is generally found that theyhave a high clearance. A high clearance of a therapeutic agent isinconvenient in cases where it is desired to maintain a high blood levelthereof over a prolonged period of time since repeated administrationswill then be necessary. Examples of peptides which have a high clearanceare: ACTH, corticotropin-releasing factor, angiotensin, calcitonin,insulin, glucagon, glucagon-like peptide-1, glucagon-like peptide-2,insulin-like growth factor-1, insulin-like growth factor-2, gastricinhibitory peptide, growth hormone-releasing factor, pituitary adenylatecyclase activating peptide, secretin, enterogastrin, somatostatin,somatotropin, somatomedin, parathyroid hormone, thrombopoietin,erythropoietin, hypothalamic releasing factors, prolactin, thyroidstimulating hormones, endorphins, enkephalins, vasopressin, oxytocin,opiods and analogues thereof, superoxide dismutase, interferon,asparaginase, arginase, arginine deaminase, adenosine deaminase andribonuclease. In some cases it is possible to influence the releaseprofile of peptides by applying suitable pharmaceutical compositions,but this approach has various shortcomings and is not generallyapplicable.

The hormones regulating insulin secretion belong to the so-calledenteroinsular axis, designating a group of hormones, released from thegastrointestinal mucosa in response to the presence and absorption ofnutrients in the gut, which promote an early and potentiated release ofinsulin. The enhancing effect on insulin secretion, the so-calledincretin effect, is probably essential for a normal glucose tolerance.Many of the gastrointestinal hormones, including gastrin and secretin(cholecystokinin is not insulinotropic in man), are insulinotropic, butthe only physiologically important ones, those that are responsible forthe incretin effect, are the glucose-dependent insulinotropicpolypeptide, GIP, and glucagon-like peptide-1 (GLP-1). Because of itsinsulinotropic effect, GIP, isolated in 1973 (1) immediately attractedconsiderable interest among diabetologists. However, numerousinvestigations carried out during the following years clearly indicatedthat a defective secretion of GIP was not involved in the pathogenesisof insulin dependent diabetes mellitus (IDDM) or non insulin-dependentdiabetes mellitus (NIDDM) (2). Furthermore, as an insulinotropichormone, GIP was found to be almost ineffective in NIDDM (2). The otherincretin hormone, GLP-1 is the most potent insulinotropic substanceknown (3). Unlike GIP, it is surprisingly effective in stimulatinginsulin secretion in NIDDM patients. In addition, and in contrast to theother insulinotropic hormones (perhaps with the exception of secretin)it also potently inhibits glucagon secretion. Because of these actionsit has pronounced blood glucose lowering effects particularly inpatients with NIDDM.

GLP-1, a product of the proglucagon (4), is one of the youngest membersof the secretin-VIP family of peptides, but is already established as animportant gut hormone with regulatory function in glucose metabolism andgastrointestinal secretion and metabolism (5). The glucagon gene isprocessed differently in the pancreas and in the intestine. In thepancreas (9), the processing leads to the formation and parallelsecretion of 1) glucagon itself, occupying positions 33-61 ofproglucagon (PG); 2) an N-terminal peptide of 30 amino acids (PG (1-30))often called glicentin-related pancreatic peptide, GRPP (10, 11); 3) ahexapeptide corresponding to PG (64-69); 4) and, finally, the so-calledmajor proglucagon fragment (PG (72-158)), in which the two glucagon-likesequences are buried (9). Glucagon seems to be the only biologicallyactive product. In contrast, in the intestinal mucosa, it is glucagonthat is buried in a larger molecule, while the two glucagon-likepeptides are formed separately (8). The following products are formedand secreted in parallel: 1) glicentin, corresponding to PG (1-69), withthe glucagon sequence occupying residues Nos. 33-61 (12); 2)GLP-1(7-36)amide (PG (78-107))amide (13), not as originally believed PG(72-107)amide or 108, which is inactive). Small amounts of C-terminallyglycine-extended but equally bioactive GLP-1(7-37), (PG (78-108)) arealso formed (14); 3) intervening peptide-2 (PG (111-122)amide) (15); and4) GLP-2 (PG (126-158)) (15, 16). A fraction of glicentin is cleavedfurther into GRPP (PG (1-30)) and oxyntomodulin (PG (33-69)) (17, 18).Of these peptides, GLP-1, has the most conspicuous biologicalactivities.

Being secreted in parallel with glicentin/enteroglucagon, it followsthat the many studies of enteroglucagon secretion (6, 7) to some extentalso apply to GLP-1 secretion, but GLP-1 is metabolised more quicklywith a plasma half-life in humans of 2 min (19). Carbohydrate orfat-rich meals stimulate secretion (20), presumably as a result ofdirect interaction of yet unabsorbed nutrients with the microvilli ofthe open-type L-cells of the gut mucosa. Endocrine or neural mechanismspromoting GLP-1 secretion may exist but have not yet been demonstratedin humans.

The incretin function of GLP-1(29-31) has been clearly illustrated inexperiments with the GLP-1 receptor antagonist, exendin 9-39, whichdramatically reduces the incretin effect elicited by oral glucose inrats (21, 22). The hormone interacts directly with the β-cells via theGLP-1 receptor (23) which belongs to the glucagon/VIP/calcitonin familyof G-protein-coupled 7-transmembrane spanning receptors. The importanceof the GLP-1 receptor in regulating insulin secretion was illustrated inrecent experiments in which a targeted disruption of the GLP-1 receptorgene was carried out in mice. Animals homozygous for the disruption hadgreatly deteriorated glucose tolerance and fasting hyperglycaemia, andeven heterozygous animals were glucose intolerant (24). The signaltransduction mechanism (25) primarily involves activation of adenylatecyclase, but elevations of intracellular Ca²⁺ are also essential (25,26). The action of the hormone is best described as a potentiation ofglucose stimulated insulin release (25), but the mechanism that couplesglucose and GLP-1 stimulation is not known. It may involve acalcium-induced calcium release (26, 27). As already mentioned, theinsulinotropic action of GLP-1 is preserved in diabetic β-cells. Therelation of the latter to its ability to convey “glucose competence” toisolated insulin-secreting cells (26, 28), which respond poorly toglucose or GLP-1 alone, but fully to a combination of the two, is alsonot known. Equally importantly, however, the hormone also potentlyinhibits glucagon secretion (29). The mechanism is not known, but seemsto be paracrine, via neighbouring insulin or somatostatin cells (25).Also the glucagonostatic action is glucose-dependent, so that theinhibitory effect decreases as blood glucose decreases. Because of thisdual effect, if the plasma GLP-1 concentrations increase either byincreased secretion or by exogenous infusion the molar ratio of insulinto glucagon in the blood that reaches the liver via the portalcirculation is greatly increased, whereby hepatic glucose productiondecreases (30). As a result blood glucose concentrations decrease.Because of the glucose dependency of the insulinotropic andglucagonostatic actions, the glucose lowering effect is self-limiting,and the hormone, therefore, does not cause hypoglycaemia regardless ofdose (31). The effects are preserved in patients with diabetes mellitus(32), in whom infusions of slightly supraphysiological doses of GLP-1may completely normalise blood glucose values in spite of poor metaboliccontrol and secondary failure to sulphonylurea (33). The importance ofthe glucagonostatic effect is illustrated by the finding that GLP-1 alsolowers blood glucose in type-1 diabetic patients without residual O-cellsecretory capacity (34).

In addition to its effects on the pancreatic islets, GLP-1 has powerfulactions on the gastrointestinal tract. Infused in physiological amounts,GLP-1 potently inhibits pentagastrin-induced as well as meal-inducedgastric acid secretion (35, 36). It also inhibits gastric emptying rateand pancreatic enzyme secretion (36). Similar inhibitory effects ongastric and pancreatic secretion and motility may be elicited in humansupon perfusion of the ileum with carbohydrate- or lipid-containingsolutions (37, 38). Concomitantly, GLP-1 secretion is greatlystimulated, and it has been speculated that GLP-1 may be at least partlyresponsible for this so-called “ileal-brake” effect (38). In fact,recent studies suggest that, physiologically, the ileal-brake effects ofGLP-1 may be more important than its effects on the pancreatic islets.Thus, in dose response studies GLP-1 influences gastric emptying rate atinfusion rates at least as low as those required to influence isletsecretion (39).

GLP-1 seems to have an effect on food intake. Intraventricularadministration of GLP-1 profoundly inhibits food intake in rats (40,42). This effect seems to be highly specific. Thus, N-terminallyextended GLP-1 (PG 72-107)amide is inactive and appropriate doses of theGLP-1 antagonist, exendin 9-39, abolish the effects of GLP-1 (41).Acute, peripheral administration of GLP-1 does not inhibit food intakeacutely in rats (41, 42). However, it remains possible that GLP-1secreted from the intestinal L-cells may also act as a satiety signal.

Not only the insulinotropic effects but also the effects of GLP-1 on thegastrointestinal tract are preserved in diabetic patients (43), and mayhelp curtailing meal-induced glucose excursions, but, more importantly,may also influence food intake. Administered intravenously, continuouslyfor one week, GLP-1 at 4 ng/kg/min has been demonstrated to dramaticallyimprove glycaemic control in NIDDM patients without significant sideeffects (44). The peptide is fully active after subcutaneousadministration (45), but is rapidly degraded mainly due to degradationby dipeptidyl peptidase IV-like enzymes (46, 47).

The amino acid sequence of GLP-1 is given i.a. by Schmidt et al.(Diabetologia 28 704-707 (1985). Although the interestingpharmacological properties of GLP-1(7-37) and analogues thereof haveattracted much attention in recent years only little is known about thestructure of these molecules. The secondary structure of GLP-1 inmicelles has been described by Thorton et al. (Biochemistry 33 3532-3539(1994)), but in normal solution, GLP-1 is considered a very flexiblemolecule. Surprisingly, we found that derivatisation of this relativelysmall and very flexible molecule resulted in compounds whose plasmaprofile were highly protracted and still had retained activity.

GLP-1 and analogues of GLP-1 and fragments thereof are potentiallyuseful i.a. in the treatment of type 1 and type 2 diabetes. However, thehigh clearance limits the usefulness of these compounds, and thus therestill is a need for improvements in this field. Accordingly, it is oneobject of the present invention to provide derivatives of GLP-1 andanalogues thereof which have a protracted profile of action relative toGLP-1(7-37). It is a further object of the invention to providederivatives of GLP-1 and analogues thereof which have a lower clearancethan GLP-1(7-37). It is a further object of the invention to provide apharmaceutical composition comprising a compound according to theinvention and to use a compound of the invention to provide such acomposition. Also, it is an object of the present invention to provide amethod of treating insulin dependent and non-insulin dependent diabetesmellitus.

REFERENCES

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SUMMARY OF THE INVENTION

Human GLP-1 is a 37 amino acid residue peptide originating frompreproglucagon which is synthesised i.a. in the L-cells in the distalileum, in the pancreas and in the brain. Processing of preproglucagon togive GLP-1(7-36)amide, GLP-1(7-37) and GLP-2 occurs mainly in theL-cells. A simple system is used to describe fragments and analogues ofthis peptide. Thus, for example, Gly⁸-GLP-1(7-37) designates a fragmentof GLP-1 formally derived from GLP-1 by deleting the amino acid residuesNos. 1 to 6 and substituting the naturally occurring amino acid residuein position 8 (Ala) by Gly. Similarly,Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-37) designates GLP-1(7-37) whereinthe ε-amino group of the Lys residue in position 34 has beentetradecanoylated. Where reference in this text is made to C-terminallyextended GLP-1 analogues, the amino acid residue in position 38 is Argunless otherwise indicated, the optional amino acid residue in position39 is also Arg unless otherwise indicated and the optional amino acidresidue in position 40 is Asp unless otherwise indicated. Also, if aC-terminally extended analogue extends to position 41, 42, 43, 44 or 45,the amino acid sequence of this extension is as in the correspondingsequence in human preproglucagon unless otherwise indicated.

In its broadest aspect, the present invention relates to derivatives ofGLP-1 and analogues thereof. The derivatives according to the inventionhave interesting pharmacological properties, in particular they have amore protracted profile of action than the parent peptides.

In the present text, the designation “an analogue” is used to designatea peptide wherein one or more amino acid residues of the parent peptidehave been substituted by another amino acid residue and/or wherein oneor more amino acid residues of the parent peptide have been deletedand/or wherein one or more amino acid residues have been added to theparent peptide. Such addition can take place either at the N-terminalend or at the C-terminal end of the parent peptide or both.

The term “derivative” is used in the present text to designate a peptidein which one or more of the amino acid residues of the parent peptidehave been chemically modified, e.g. by alkylation, acylation, esterformation or amide formation.

The term “a GLP-1 derivative” is used in the present text to designate aderivative of GLP-1 or an analogue thereof. In the present text, theparent peptide from which such a derivative is formally derived is insome places referred to as the “GLP-1 moiety” of the derivative.

In a preferred embodiment, the present invention relates to a GLP-1derivative wherein at least one amino acid residue of the parent peptidehas a lipophilic substituent attached with the proviso that if only onelipophilic substituent is present and this substituent is attached tothe N-terminal or to the C-terminal amino acid residue of the parentpeptide then this substituent is an alkyl group or a group which has anω-carboxylic acid group.

In another preferred embodiment, the present invention relates to aGLP-1 derivative having only one lipophilic substituent.

In another preferred embodiment, the present invention relates to aGLP-1 derivative having only one lipophilic substituent whichsubstituent is an alkyl group or a group which has an (O-carboxylic acidgroup and is attached to the N-terminal amino acid residue of the parentpeptide.

In another preferred embodiment, the present invention relates to aGLP-1 derivative having only one lipophilic substituent whichsubstituent is an alkyl group or a group which has an ω-carboxylic acidgroup and is attached to the C-terminal amino acid residue of the parentpeptide.

In another preferred embodiment, the present invention relates to aGLP-1 derivative having only one lipophilic substituent whichsubstituent can be attached to any one amino acid residue which is notthe N-terminal or C-terminal amino acid residue of the parent peptide.

In another preferred embodiment, the present invention relates to aGLP-1 derivative wherein two lipophilic substituents are present.

In another preferred embodiment, the present invention relates to aGLP-1 derivative wherein two lipophilic substituents are present, onebeing attached to the N-terminal amino acid residue while the other isattached to the C-terminal amino acid residue.

In another preferred embodiment, the present invention relates to aGLP-1 derivative wherein two lipophilic-substituents are present, onebeing attached to the N-terminal amino acid residue while the other isattached to an amino acid residue which is not N-terminal or theC-terminal amino acid residue.

In another preferred embodiment, the present invention relates to aGLP-1 derivative wherein two lipophilic substituents are present, onebeing attached to the C-terminal amino acid residue while the other isattached to an amino acid residue which is not the N-terminal or theC-terminal amino acid residue.

In a further preferred embodiment, the present invention relates to aderivative of GLP-1(7-C), wherein C is selected from the groupcomprising 38, 39, 40, 41, 42, 43, 44 and 45 which derivative has justone lipophilic substituent which is attached to the C-terminal aminoacid residue of the parent peptide.

In a further preferred embodiment, the present invention relates to aGLP-1 derivative, being a derivative of GLP-1(7-C), wherein C is 35 or36 which derivative has just one lipophilic substituent which isattached to the C-terminal amino acid residue.

In a further preferred embodiment, the present invention relates to aGLP-1 derivative wherein the lipophilic substituent comprises from 4 to40 carbon atoms, more preferred from 8 to 25 carbon atoms.

In a further preferred embodiment, the present invention relates to aGLP-1 derivative wherein a lipophilic substituent is attached to anamino acid residue in such a way that a carboxyl group of the lipophilicsubstituent forms an amide bond with an amino group of the amino acidresidue.

In a further preferred embodiment, the present invention relates to aGLP-1 derivative wherein a lipophilic substituent is attached to anamino acid residue in such a way that an amino group of the lipophilicsubstituent forms an amide bond with a carboxyl group of the amino acidresidue.

In a further preferred embodiment, the present invention relates to aGLP-1 derivative wherein a lipophilic substituent is attached to theparent peptide by means of a spacer.

In a further preferred embodiment, the present invention relates to aGLP-1 derivative wherein a lipophilic substituent—optionally via aspacer—is attached to the ε-amino group of a Lys residue contained inthe parent peptide.

In a further preferred embodiment, the present invention relates to aGLP-1 derivative wherein a lipophilic substituent is attached to theparent peptide by means of a spacer which is an unbranched alkaneα,ω-dicarboxylic acid group having from 1 to 7 methylene groups,preferably two methylene groups which spacer forms a bridge between anamino group of the parent peptide and an amino group of the lipophilicsubstituent.

In a further preferred embodiment, the present invention relates to aGLP-1 derivative wherein a lipophilic substituent is attached to theparent peptide by means of a spacer which is an amino acid residueexcept Cys, or a dipeptide such as Gly-Lys. In the present text, theexpression “a dipeptide such as Gly-Lys” is used to designate adipeptide wherein the C-terminal amino acid residue is Lys, His or Trp,preferably Lys, and wherein the N-terminal amino acid residue isselected from the group comprising Ala, Arg, Asp, Asn, Gly, Glu, Gln,Ile, Leu, Val, Phe and Pro.

In a further preferred embodiment, the present invention relates to aGLP-1 derivative wherein a lipophilic substituent is attached to theparent peptide by means of a spacer which is an amino acid residueexcept Cys, or is a dipeptide such as Gly-Lys and wherein a carboxylgroup of the parent peptide forms an amide bond with an amino group of aLys residue or a dipeptide containing a Lys residue, and the other aminogroup of the Lys residue or a dipeptide containing a Lys residue formsan amide bond with a carboxyl group of the lipophilic substituent.

In a further preferred embodiment, the present invention relates to aGLP-1 derivative wherein a lipophilic substituent is attached to theparent peptide by means of a spacer which is an amino acid residueexcept Cys, or is a dipeptide such as Gly-Lys and wherein an amino groupof the parent peptide forms an amide bond with a carboxylic group of theamino acid residue or dipeptide spacer, and an amino group of the aminoacid residue or dipeptide spacer forms an amide bond with a carboxylgroup of the lipophilic substituent.

In a further preferred embodiment, the present invention relates to aGLP-1 derivative wherein a lipophilic substituent is attached to theparent peptide by means of a spacer which is an amino acid residueexcept Cys, or is a dipeptide such as Gly-Lys and wherein a carboxylgroup of the parent peptide forms an amide bond with an amino group ofthe amino acid residue spacer or dipeptide spacer, and the carboxylgroup of the amino acid residue spacer or dipeptide spacer forms anamide bond with an amino group of the lipophilic substituent.

In a further preferred embodiment, the present invention relates to aGLP-1 derivative wherein a lipophilic substituent is attached to theparent peptide by means of a spacer which is an amino acid residueexcept Cys, or is a dipeptide such as Gly-Lys, and wherein a carboxylgroup of the parent peptide forms an amide bond with an amino group of aspacer which is Asp or Glu, or a dipeptide spacer containing an Asp orGlu residue, and a carboxyl group of the spacer forms an amide bond withan amino group of the lipophilic substituent.

In a further preferred embodiment, the present invention relates to aGLP-1 derivative having a lipophilic substituent which comprises apartially or completely hydrogenated cyclopentanophenathrene skeleton.

In a further preferred embodiment, the present invention relates to aGLP-1 derivative having a lipophilic substituent which is astraight-chain or branched alkyl group.

In a further preferred embodiment, the present invention relates to aGLP-1 derivative having a lipophilic substituent which is the acyl groupof a straight-chain or branched fatty acid.

In a further preferred embodiment, the present invention relates to aGLP-1 derivative having a lipophilic substituent which is an acyl groupselected from the group comprising CH₃(CH₂)_(n)CO—, wherein n is aninteger from 4 to 38, preferably an integer from 4 to 24, more preferredselected from the group comprising CH₃(CH₂)₆CO—, CH₃(CH₂)₈CO—,CH₃(CH₂)₁₀CO—, CH₃(CH₂)₁₂CO—, CH₃(CH₂)₁₄CO—, CH₃(CH₂)₁₆CO—,CH₃(CH₂)₁₈CO—, CH₃(CH₂)₂₀CO— and CH₃(CH₂)₂₂CO—.

In a further preferred embodiment, the present invention relates to aGLP-1 derivative having a lipophilic substituent which is an acyl groupof a straight-chain or branched alkane α,ω-dicarboxylic acid.

In a further preferred embodiment, the present invention relates to aGLP-1 derivative having a lipophilic substituent which is an acyl groupselected from the group comprising HOOC(CH₂)_(m)CO—, wherein m is aninteger from 4 to 38, preferably an integer from 4 to 24, more preferredselected from the group comprising HOOC(CH₂)₁₄CO—, HOOC(CH₂)₁₆CO—,HOOC(CH₂)₁₈CO—, HOOC(CH₂)₂₀CO— and HOOC(CH₂)₂₂CO—.

In a further preferred embodiment, the present invention relates to aGLP-1 derivative having a lipophilic substituent which is a group of theformula CH₃(CH₂)_(p)((CH₂)_(q)COOH)CHNH—CO(CH₂)₂CO—, wherein p and q areintegers and p+q is an integer of from 8 to 33, preferably from 12 to28.

In a further preferred embodiment, the present invention relates to aGLP-1 derivative having a lipophilic substituent which is a group of theformula CH₃(CH₂)_(r)CO—NHCH(COOH)(CH₂)₂CO—, wherein r is an integer offrom 10 to 24.

In a further preferred embodiment, the present invention relates to aGLP-1 derivative having a lipophilic substituent which is a group of theformula CH₃(CH₂)_(s)CO—NHCH((CH₂)₂COOH)CO—, wherein s is an integer offrom 8 to 24.

In a further preferred embodiment, the present invention relates to aGLP-1 derivative having a lipophilic substituent which is a group of theformula COOH(CH₂)_(t)CO— wherein t is an integer of from 8 to 24.

In a further preferred embodiment, the present invention relates to aGLP-1 derivative having a lipophilic substituent which is a group of theformula —NHCH(COOH)(CH₂)₄NH—CO(CH₂)_(u)CH₃, wherein u is an integer offrom 8 to 18.

In a further preferred embodiment, the present invention relates to aGLP-1 derivative having a lipophilic substituent which is a group of theformula —NHCH(COOH)(CH₂)₄NH—COCH((CH₂)₂COOH)NH—CO(CH₂)_(w)CH₃, wherein wis an integer of from 10 to 16.

In a further preferred embodiment, the present invention relates to aGLP-1 derivative having a lipophilic substituent which is a group of theformula —NHCH(COOH)(CH₂)₄NH—CO(CH₂)₂CH(COOH)NH—CO(CH₂)_(x)CH₃, wherein xis an integer of from 10 to 16.

In a further preferred embodiment, the present invention relates to aGLP-1 derivative having a lipophilic substituent which is a group of theformula —NHCH(COOH)(CH₂)₄NH—CO(CH₂)₂CH(COOH)NHCO(CH₂)_(y)CH₃, wherein yis zero or an integer of from 1 to 22.

In a further preferred embodiment, the present invention relates to aGLP-1 derivative having a lipophilic substituent which can be negativelycharged. Such a lipophilic substituent can for example be a substituentwhich has a carboxyl group.

In a further preferred embodiment, the present invention relates to aGLP-1 derivative the parent peptide of which is selected from the groupcomprising GLP-1(1-45) or an analogue thereof.

In a further preferred embodiment, the present invention relates to aGLP-1 derivative derived from a GLP-1 fragment selected from the groupcomprising GLP-1(7-35), GLP-1(7-36), GLP-1(7-36)amide, GLP-1(7-37),GLP-1(7-38), GLP-1(7-39), GLP-1(7-40) and GLP-1(7-41) or an analoguethereof.

In a further preferred embodiment, the present invention relates to aGLP-1 analogue derived from a GLP-1 analogue selected from the groupcomprising GLP-1(1-35), GLP-1(1-36), GLP-1(1-36)amide, GLP-1(1-37),GLP-1(1-38), GLP-1(1-39), GLP-1(1-40) and GLP-1(1-41) or an analoguethereof.

In a further preferred embodiment, the present invention relates to aGLP-1 derivative wherein the designation analogue comprises derivativeswherein a total of up to fifteen, preferably up to ten amino acidresidues have been exchanged with any α-amino acid residue.

In a further preferred embodiment, the present invention relates to aGLP-1 derivative wherein the designation analogue comprises derivativeswherein a total of up to fifteen, preferably up to ten amino acidresidues have been exchanged with any α-amino acid residue which can becoded for by the genetic code.

In a further preferred embodiment, the present invention relates to aGLP-1 derivative wherein the designation analogue comprises derivativeswherein a total of up to six amino acid residues have been exchangedwith another α-amino acid residue which can be coded for by the geneticcode.

In a further preferred embodiment, the present invention relates to aGLP-1(A-B) derivative wherein A is an integer from 1 to 7 and B is aninteger from 38 to 45 or an analogue thereof comprising one lipophilicsubstituent attached to the C-terminal amino acid residue and,optionally, a second lipophilic substituent attached to one of the otheramino acid residues.

In a further preferred embodiment, a parent peptide for a derivativeaccording to the invention is selected from the group comprisingArg²⁶-GLP-1(7-37); Arg³⁴-GLP-1(7-37); Lys³⁶-GLP-1(7-37);Arg^(26,34)Lys³⁶-GLP-1(7-37); Arg^(26,34)Lys³⁸GLP-1(7-38);Arg^(26,34)Lys³⁹-GLP-1(7-39); Arg^(26,34)Lys⁴⁰-GLP-1(7-40);Arg²⁶Lys³⁶-GLP-1(7-37); Arg³⁴Lys³⁶-GLP-1(7-37); Arg²⁶Lys³⁹-GLP-1(7-39);Arg³⁴Lys⁴⁰-GLP-1(7-40); Arg^(26,34)Lys^(36,39)-GLP-1(7-39);Arg^(26,34)Lys^(36,40)-GLP-1(7-40); Gly⁸Arg26-GLP-1(7-37);Gly⁸Arg³⁴-GLP-1(7-37); Gly⁸Lys³⁶GLP-1(7-37);Gly⁸Arg^(26,34)Lys³⁶-GLP-1(7-37); Gly⁸Arg^(26,34)Lys³⁹-GLP-1(7-39);Gly⁸Arg^(26,34)Lys⁴⁰-GLP-1(7-40); Gly⁸Arg²⁶Lys³⁶-GLP-1(7-37);Gly⁸Arg³⁴Lys³⁶-GLP-1(7-37); Gly⁸Arg²⁶Lys³⁹-GLP-1(7-39);Gly⁸Arg³⁴Lys⁴⁰-GLP-1(7-40); Gly⁸Arg^(26,34)Lys^(36,39)-GLP-1(7-39) andGly⁸Arg^(26,34)Lys^(36,40)-GLP-1(7-40).

In a further preferred embodiment, a parent peptide for a derivativeaccording to the invention is selected from the group comprisingArg^(26,34)Lys³⁸GLP-1(7-38); Arg^(26,34)Lys³⁹GLP-1(7-39);Arg^(26,34)Lys⁴⁰GLP-1(7-40); Arg^(26,34)Lys⁴¹GLP-1(7-41);Arg^(26,34)Lys⁴²GLP-1(7-42); Arg^(26,34)Lys⁴³GLP-1(7-43);Arg^(26,34)Lys⁴⁴GLP-1(7-44); Arg^(26,34)Lys⁴⁵GLP-1(7-45);Arg^(26,34)Lys³⁸GLP-1(1-38); Arg^(26,34)Lys³⁹GLP-1(1-39);Arg^(26,34)Lys⁴⁰GLP-1(1-40); Arg^(26,34)Lys⁴¹GLP-1(1-41);Arg^(26,34)Lys⁴²GLP-1(1-42); Arg^(26,34)Lys⁴³GLP-1(1-43);Arg^(26,34)Lys⁴⁴GLP-1(1-44); Arg^(26,34)Lys⁴⁵GLP-1(1-45);Arg^(26,34)Lys³⁸GLP-1(2-38); Arg^(26,34)Lys³⁹GLP-1(2-39);Arg^(26,34)Lys⁴⁰GLP-1(2-40); Arg^(26,34)Lys⁴¹GLP-1(2-41);Arg^(26,34)Lys⁴²GLP-1(2-42); Arg^(26,34)Lys⁴³GLP-1(2-43);Arg^(26,34)Lys⁴⁴GLP-1(2-44); Arg^(23,34)Lys⁴⁵GLP-1(2-45);Arg^(26,34)Lys³⁸GLP-1(3-38); Arg^(26,34)Lys³⁹GLP-1(3-39);Arg^(26,34)Lys⁴⁰GLP-1(3-40); Arg^(26,34)Lys⁴¹GLP-1(3-41);Arg^(26,34)Lys⁴²GLP-1(3-42); Arg^(26,34)Lys⁴³GLP-1(3-43);Arg^(26,34)Lys⁴⁴GLP-1(3-44); Arg^(26,34)Lys⁴⁵GLP-1(3-45);Arg^(26,34)Lys³⁸GLP-1(4-38); Arg^(26,34)Lys³⁹GLP-1(4-39);Arg^(26,34)Lys⁴⁰GLP-1(4-40); Arg^(26,34)Lys⁴¹GLP-1(4-41);Arg^(26,34)Lys⁴²GLP-1(4-42); Arg^(26,34)Lys⁴³GLP-1(4-43);Arg^(26,34)Lys⁴⁴GLP-1(4-44); Arg^(26,34)Lys⁴⁵GLP-1(4-45);Arg^(26,34)Lys³⁸GLP-1(5-38); Arg^(26,34)Lys³⁹GLP-1(5-39);Arg^(26,34)Lys⁴⁰GLP-1(5-40); Arg^(26,34)Lys⁴¹GLP-1(5-41);Arg^(26,34)Lys⁴²GLP-1(5-42); Arg^(26,34)Lys⁴³GLP-1(5-43);Arg^(26,34)Lys⁴⁴GLP-1(5-44); Arg^(26,34)Lys⁴⁵GLP-1(5-45);Arg^(26,34)Lys³⁸GLP-1(6-38); Arg^(26,34)Lys³⁹GLP-1(6-39);Arg^(26,34)Lys⁴⁰GLP-1(6-40); Arg^(26,34)Lys⁴¹GLP-1(6-41);Arg^(26,34)Lys⁴²GLP-1(6-42); Arg^(26,34)Lys⁴³GLP-1(6-43);Arg^(26,34)Lys⁴⁴GLP-1(6-44); Arg^(26,34)Lys⁴⁵GLP-1(6-45);Arg²⁶Lys³⁸GLP-1(1-38); Arg³⁴Lys³⁸GLP-1(1-38);Arg^(26,34)Lys^(36,38)GLP-1(1-38); Arg²⁶Lys³⁸GLP-1(7-38);Arg³⁴Lys³⁸GLP-1(7-38); Arg^(26,34)Lys^(36,38)GLP-1(7-38);Arg^(26,34)Lys³⁸GLP-1(7-38); Arg²⁶Lys³⁹GLP-1(1-39);Arg³⁴Lys³⁹GLP-1(1-39); Arg^(26,34)Lys^(36,39)GLP-1(1-39);Arg²⁶Lys³⁹GLP-1(7-39); Arg³⁴Lys³⁹GLP-1(7-39) andArg^(26,34)Lys^(36,39)GLP-1(7-39).

In a further preferred embodiment, the present invention relates to aGLP-1 derivative wherein the parent peptide is selected from the groupcomprising Arg²⁶-GLP-1(7-37), Arg³⁴-GLP-1(7-37), Lys³⁶GLP-1(7-37),Arg^(26,34)Lys³⁶-GLP-1(7-37), Arg²⁶Lys³⁶-GLP-1(7-37),Arg³⁴Lys³⁶-GLP-1(7-37), Gly⁸Arg²⁶-GLP-1(7-37), Gly⁸Arg³⁴-GLP-1(7-37),Gly⁸Lys³⁶-GLP-1(7-37), Gly⁸Arg^(26,34)Lys³⁶-GLP-1(7-37),Gly⁸Arg²⁶Lys³⁶-GLP-1(7-37) and Gly⁸Arg³⁴Lys³⁶-GLP-1(7-37).

In a further preferred embodiment, the present invention relates to aGLP-1 derivative wherein the parent peptide is selected from the groupcomprising Arg²⁶Lys³⁸-GLP-1(7-38), Arg^(26,34)Lys³⁸-GLP-1(7-38),Arg^(26,34)Lys^(36,38)-GLP-1(7-38), Gly⁸Arg²⁶Lys³⁸-GLP-1(7-38) andGly⁸Arg^(26,34)Lys^(36,38)-GLP-1(7-38).

In a further preferred embodiment, the present invention relates to aGLP-1 derivative wherein the parent peptide is selected from the groupcomprising Arg²⁶Lys³⁹-GLP-1(7-39), Arg^(26,34)Lys^(36,39)-GLP-1(7-39),Gly⁸Arg²⁶Lys³⁹-GLP-1(7-39) and Gly⁸Arg^(26,34)Lys^(36,39)-GLP-1(7-39).

In a further preferred embodiment, the present invention relates to aGLP-1 derivative wherein the parent peptide is selected from the groupcomprising Arg³⁴Lys⁴⁰-GLP-1(7-40), Arg^(26,34)Lys^(36,40)-GLP-1(7-40),Gly⁸Arg³⁴Lys⁴⁰-GLP-1(7-40) and Gly⁸Arg^(26,34)Lys^(36,40)-GLP-1(7-40).

In a further preferred embodiment, the present invention relates to aGLP-1 derivative which is selected from the group comprising:

Lys²⁶(N^(ε)-tetradecanoyl)-GLP-1(7-37);Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-37);Lys^(26,34)-bis(N^(ε)-tetradecanoyl)-GLP-1(7-37);Gly⁸Lys²⁶(N^(ε)-tetradecanoyl)-GLP-1(7-37);Gly⁸Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-37);Gly⁸Lys^(26,34)-bis(N^(ε)-tetradecanoyl)-GLP-1(7-37);Arg²⁶Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-37);Lys²⁶(N^(ε)-tetradecanoyl)-GLP-1(7-38);Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-38);Lys^(26,34)(N^(ε)-tetradecanoyl)-GLP-1(7-38);Gly⁸Lys²⁶(N^(ε)-tetradecanoyl)-GLP-1(7-38);Gly⁸Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-38);Gly⁸Lys^(26,34)-bis(N^(ε)-tetradecanoyl)-GLP-1(7-38);Arg²⁶Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-38);Lys²⁶(N^(ε)-tetradecanoyl)-GLP-1(7-39);Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-39);Lys^(26,34)-bis(N^(ε)-tetradecanoyl)-GLP-1(7-39);Gly⁸Lys²⁶(N^(ε)-tetradecanoyl)-GLP-1(7-39);Gly⁸Lys³⁴(N^(ε)-tetradecanoyl)-GLP-2 (7-39);Gly⁸Lys^(26,34)-bis(N^(ε)-tetradecanoyl)-GLP-1(7-39);Arg²⁶Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-39);Lys²⁶(N^(ε)-tetradecanoyl)-GLP-1(7-40);Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-40);Lys^(26,34)-bis(N^(ε)-tetradecanoyl)-GLP-1(7-40);Gly⁸Lys²⁶(N^(ε)-tetradecanoyl)-GLP-1(7-40);Gly⁸Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-40);Gly⁸Lys^(26,34)-bis(N^(ε)-tetradecanoyl)-GLP-1(7-40);Arg²⁶Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-40);Lys²⁶(N^(ε)-tetradecanoyl)-GLP-1(7-36);Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-36);Lys^(26,34)-bis(N^(ε)-tetradecanoyl)-GLP-1(7-36);Gly⁸Lys²⁶(N^(ε)-tetradecanoyl)-GLP-1(7-36);Gly⁸Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-36);Gly⁸Lys^(26,34)-bis(N^(ε)-tetradecanoyl)-GLP-1(7-36);Arg²⁶Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-36);Lys²⁶(N^(ε)-tetradecanoyl)-GLP-1(7-35);Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-35);Lys^(26,34)-bis(N^(ε)-tetradecanoyl)-GLP-1(7-35);Gly⁸Lys²⁶(N^(ε)-tetradecanoyl)-GLP-1(7-35);Gly⁸Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-35);Gly⁸Lys^(26,34)-bis(N^(ε)-tetradecanoyl)-GLP-1(7-35);Arg²⁶Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-35);Lys²⁶(N^(ε)-tetradecanoyl)-GLP-1(7-36)amide;Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-36)amide;Lys^(26,34)-bis(N^(ε)-tetradecanoyl)-GLP-1(7-36)amide;Gly⁸Lys²⁶(N^(ε)-tetradecanoyl)-GLP-1(7-36)amide;Gly⁸Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-36)amide;Gly⁸Lys^(26,34)-bis(N^(ε)-tetradecanoyl)-GLP-1(7-36)amide;Arg²⁶Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-36)amide;Gly⁸Arg²⁶Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-37);Lys²⁶(N^(ε)-tetradecanoyl)Arg³⁴-GLP-1(7-37);Gly⁸Lys²⁶(N^(ε)-tetradecanoyl)Arg³⁴-GLP-1(7-37);Arg^(26,34)Lys³⁶(N^(ε)-tetradecanoyl)-GLP-1(7-37);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-tetradecanoyl)-GLP-1(7-37);Gly⁸Arg²⁶Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-38);Lys²⁶(N^(ε)-tetradecanoyl)Arg³⁴-GLP-1(7-38);Gly⁸Lys²⁶(N^(ε)-tetradecanoyl)Arg³⁴-GLP-1(7-38);Arg^(26,34)Lys³⁶(N^(ε)-tetradecanoyl)-GLP-1(7-38);Arg^(26,34)Lys³⁸(N^(ε)-tetradecanoyl)-GLP-1(7-38);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-tetradecanoyl)-GLP-1(7-38);Gly⁸Arg²⁶Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-38);Lys²⁶(N^(ε)-tetradecanoyl)Arg³⁴-GLP-1(7-39);Gly⁸Lys²⁶(N^(ε)-tetradecanoyl)Arg³⁴-GLP-1(7-39);Arg^(26,34)Lys³⁶(N^(ε)-tetradecanoyl)-GLP-1(7-39);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-tetradecanoyl)-GLP-1(7-39);Gly⁸Arg²⁶Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-40);Lys²⁶(N^(ε)-tetradecanoyl)Arg³⁴-GLP-1(7-40);Gly⁸Lys²⁶(N^(ε)-tetradecanoyl)Arg³⁴-GLP-1(7-40);Arg^(26,34)Lys³⁶(N^(ε)-tetradecanoyl)-GLP-1(7-40);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-tetradecanoyl) GLP-1(7-40);Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-37);Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-37);Lys^(26,34)-bis(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-37);Gly⁸Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-37);Gly⁸Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-37);Gly⁸Lys^(26,34)-bis(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-37);Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-38);Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-38);Lys^(26,34)-bis(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-38);Gly⁸Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-38);Gly⁸Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-38);Gly⁸Lys^(26,34)-bis(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-38);Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-39);Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-39);Lys^(26,34)-bis(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-39);Gly⁸Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-39);Gly⁸Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-39);Gly⁸Lys^(26,34)-bis(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-39);Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-40);Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-40);Lys^(26,34)-bis(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-40);Gly⁸Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-40);Gly⁸Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-40);Gly⁸Lys^(26,34)-bis(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-40);Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-36);Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-36);Lys^(26,34)-bis(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-36);Gly⁸Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-36);Gly⁸Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-36);Gly⁸Lys^(26,34)-bis(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-36);Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-36)amide;Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-36)amide;Lys^(26,34)-bis(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-36)amide;Gly⁸Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-36)amide;Gly⁸Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-36)amide;Gly⁸Lys^(26,34)-bis(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-36)amide;Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-35);Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-35);Lys^(26,34)-bis(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-35);Gly⁸Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-35);Gly⁸Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-35);Gly⁸Lys^(26,34)-bis(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-35);Arg²⁶Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-37);Gly⁸Arg²⁶Lys³⁴(N^(ε)-(e-carboxynonadecanoyl))-GLP-1(7-37);Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))Arg³⁴-GLP-1(7-37);Gly⁸Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))Arg³⁴-GLP-1(7-37);Arg^(26,34)Lys³⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-37);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-37);Arg²⁶Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-38);Gly⁸Arg²⁶Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-38);Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))Arg³⁴-GLP-1(7-38);Gly⁸Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))Arg³⁴-GLP-1(7-38);Arg^(26,34)Lys³⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-38);Arg^(26,34)Lys³⁸(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-38);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-38);Arg²⁶Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-39);Gly⁸Arg²⁶Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-39);Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))Arg³⁴-GLP-1(7-39);Gly⁸Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))Arg³⁴-GLP-1(7-39);Arg^(26,34)Lys³⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-39);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-39);Arg²⁶Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-40);Gly⁸Arg²⁶Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-40);Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))Arg³⁴-GLP-1(7-40);Gly⁸Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))Arg³⁴-GLP-1(7-40);Arg^(26,34)Lys³⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-40);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-40);Lys²⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-37);Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-37);Lys^(26,34)-bis(N^(ε)-(7-deoxycholoyl))-GLP-1(7-37);Gly⁸Lys²⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-37);Gly⁸Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-37);Gly⁸Lys^(26,34)-bis(N^(ε)-(7-deoxycholoyl))-GLP-1(7-37);Arg²⁶Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-37);Lys²⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-38);Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-38);Lys^(26,34)-bis(N^(ε)-(7-deoxycholoyl))-GLP-1(7-38);Gly⁸Lys²⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-38);Gly⁸Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-38);Gly⁸Lys^(26,34)-bis(N^(ε)-(7-deoxycholoyl))-GLP-1(7-38);Arg²⁶Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-38);Lys²⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-39);Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-39);Lys^(26,34)-bis(N^(ε)-(7-deoxycholoyl))-GLP-1(7-39);Gly⁸Lys²⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-39);Gly⁸Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-39);Gly⁸Lys^(26,34)-bis(N^(ε)-(7-deoxycholoyl))-GLP-1(7-39);Arg²⁶Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-39);Lys²⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-40);Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-40);Lys^(26,34)-bis(N^(ε)-(7-deoxycholoyl))-GLP-1(7-40);Gly⁸Lys²⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-40);Gly⁸Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-40);Gly⁸Lys^(26,34)-bis(N^(ε)-(7-deoxycholoyl))-GLP-1(7-40);Arg²⁶Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-40);Lys²⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-36);Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-36);Lys^(26,34)-bis(N^(ε)-(7-deoxycholoyl))-GLP-1(7-36);Gly⁸Lys²⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-36);Gly⁸Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-36);Gly⁸Lys^(26,34)-bis(N^(ε)-(7-deoxycholoyl))-GLP-1(7-36);Arg²⁶Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-36);Lys²⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-35);Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-35);Lys^(26,34)-bis(N^(ε)-(7-deoxycholoyl))-GLP-1(7-35);Gly⁸Lys²⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-35);Gly⁸Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-35);Gly⁸Lys^(26,34)-bis(N^(ε)-(7-deoxycholoyl))-GLP-1(7-35);Arg²⁶Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-35);Lys²⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-36)amide;Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-36)amide;Lys^(26,34)-bis(N^(ε)-(7-deoxycholoyl))-GLP-1(7-36)amide;Gly⁸Lys²⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-36)amide;Gly⁸Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-36)amide;Gly⁸Lys^(26,34)-bis(N^(ε)-(7-deoxycholoyl))-GLP-1(7-36)amide;Arg²⁶Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-36)amide;Gly⁸Arg²⁶Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-37);Lys²⁶(N^(ε)-(7-deoxycholoyl))Arg³⁴-GLP-1(7-37);Gly⁸Lys²⁶(N^(ε)-(7-deoxycholoyl))Arg³⁴-GLP-1(7-37);Arg^(26,34)Lys³⁶(N^(ε)-7-deoxycholoyl))-GLP-1(7-37);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-37);Lys²⁶(N^(ε)-(choloyl))-GLP-1(7-37); Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-37);Lys^(26,34)-bis(N^(ε)-(choloyl))-GLP-1(7-37);Gly⁸Lys²⁶(N^(ε)-(choloyl))-GLP-1(7-37);Gly⁸Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-37);Gly⁸Lys^(26,34)-bis(N^(ε)-(choloyl))-GLP-1(7-37);Arg²⁶Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-37);Gly⁸Arg²⁶Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-38);Lys²⁶(N^(ε)-(7-deoxycholoyl))Arg³⁴-GLP-1(7-38);Gly⁸Lys²⁶(N^(ε)-(7-deoxycholoyl))Arg³⁴-GLP-1(7-38);Arg^(26,34)Lys³⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-38);Arg^(26,34)Lys³⁸(N^(ε)-(7-deoxycholoyl))-GLP-1(7-38);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-38);Lys²⁶(N^(ε)-(choloyl))-GLP-1(7-38); Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-38);Lys^(26,34)-bis(N^(ε)-(choloyl))-GLP-1(7-38);Gly⁸Lys²⁶(N^(ε)-(choloyl))-GLP-1(7-38);Gly⁸Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-38);Gly⁸Lys^(26,34)-bis(N^(ε)-(choloyl))-GLP-1(7-38);Arg²⁶Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-38);Gly⁸Arg²⁶Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-39);Lys²⁶(N^(ε)-(7-deoxycholoyl))Arg³⁴-GLP-1(7-39);Gly⁸Lys²⁶(N^(ε)-(7-deoxycholoyl))Arg³⁴-GLP-1(7-39);Arg^(26,34)Lys³⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-39);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-39);Lys²⁶(N^(ε)-(choloyl))-GLP-1(7-39); Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-39);Lys^(26,34)-bis(N^(ε)-(choloyl))-GLP-1(7-39);Gly⁸Lys²⁶(N^(ε)-(choloyl))-GLP-1(7-39);Gly⁸Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-39);Gly⁸Lys^(26,34)-bis(N^(ε)-(choloyl))-GLP-1(7-39);Arg²⁶Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-39);Gly⁸Arg²⁶Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-40);Lys²⁶(N^(ε)-(7-deoxycholoyl))Arg³⁴-GLP-1(7-40);Gly⁸Lys²⁶(N^(ε)-(7-deoxycholoyl))Arg³⁴-GLP-1(7-40);Arg^(26,34)Lys³⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-40);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-40);Lys²⁶(N^(ε)-(choloyl))-GLP-1(7-40); Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-40);Lys^(26,34)-bis(N^(ε)-(choloyl))-GLP-1(7-40);Gly⁸Lys²⁶(N^(ε)-(choloyl))-GLP-1(7-40);Gly⁸Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-40);Gly⁸Lys^(26,34)-bis(N^(ε)-(choloyl))-GLP-1(7-40);Arg²⁶Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-40);Lys²⁶(N^(ε)-(choloyl))-GLP-1(7-36); Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-36);Lys^(26,34)-bis(N^(ε)-(choloyl))-GLP-1(7-36);Gly⁸Lys²⁶(N^(ε)-(choloyl))-GLP-1(7-36);Gly⁸Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-36);Gly⁸Lys^(26,34)(N^(ε)-(choloyl)-GLP-1(7-36);Arg²⁶Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-36);Lys²⁶(N^(ε)-(choloyl))-GLP-1(7-35); Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-35);Lys^(26,34)-bis(N^(ε)-(choloyl))-GLP-1(7-35);Gly⁸Lys²⁶(N^(ε)-(choloyl))-GLP-1(7-35);Gly⁸Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-35);Gly⁸Lys^(26,34)-bis(N^(ε)-(choloyl))-GLP-1(7-35);Arg²⁶Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-35);Lys²⁶(N^(ε)-(choloyl))-GLP-1(7-36)amide;Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-36)amide;Lys^(26,34)-bis(N^(ε)-(choloyl))-GLP-1(7-36)amide;Gly⁸Lys²⁶(N^(ε)-(choloyl))-GLP-1(7-36)amide;Gly⁸Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-36)amide;Gly⁸Lys^(26,34)-bis(N^(ε)-(choloyl))-GLP-1(7-36)amide;Arg²⁶Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-36)amide;Gly⁸Arg²⁶Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-37);Lys²⁶(N^(ε)-(choloyl))Arg³⁴-GLP-1(7-37);Gly⁸Lys²⁶(N^(ε)-(choloyl))Arg³⁴-GLP-1(7-37);Arg^(26,34)Lys³⁶(N^(ε)-(choloyl))-GLP-1(7-37);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-(choloyl))-GLP-1(7-37);Lys²⁶(N^(ε)-(lithocholoyl))-GLP-1(7-37);Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-37);Lys^(26,34)-bis(N^(ε)-(lithocholoyl))-GLP-1(7-37);Gly⁸Lys²⁶(N^(ε)-(lithocholoyl))-GLP-1(7-37);Gly⁸Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-37);Gly⁸Lys^(26,34)-bis(N^(ε)-(lithocholoyl))-GLP-1(7-37);Arg²⁶Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-37);Gly⁸Arg²⁶Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-38);Lys²⁶(N^(ε)-(choloyl))Arg³⁴GLP-1(7-38);Gly⁸Lys²⁶(N^(ε)-(choloyl))Arg³⁴-GLP-1(7-38);Arg^(26,34)Lys³⁶(N^(ε)-(choloyl))-GLP-1(7-38);Arg^(26,34)Lys³⁸(N^(ε)-(choloyl))-GLP-1(7-38);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-(choloyl))-GLP-1(7-38);Lys²⁶(N^(ε)-(lithocholoyl))-GLP-1(7-38);Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-38);Lys^(26,34)-bis(N^(ε)-(lithocholoyl))-GLP-1(7-38);Gly⁸Lys²⁶(N^(ε)-(lithocholoyl))-GLP-1(7-38);Gly⁸Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-38);Gly⁸Lys^(26,34)-bis(N^(ε)-(lithocholoyl))-GLP-1(7-38);Arg²⁶Lys³⁴(N^(ε)-(choloyl)-GLP-1(7-38);Gly⁸Arg²⁶Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-39);Lys²⁶(N^(ε)-(choloyl))Arg³⁴-GLP-1(7-39);Gly⁸Lys²⁶(N^(ε)-(choloyl))Arg³⁴-GLP-1(7-39);Arg^(26,34)Lys³⁶(N^(ε)-(choloyl))-GLP-1(7-39);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-(choloyl)-GLP-1(7-39);Lys²⁶(N^(ε)-(lithocholoyl))-GLP-1(7-39);Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-39);Lys^(26,34)-bis(N^(ε)-(lithocholoyl))-GLP-1(7-39);Gly⁸Lys²⁶(N^(ε)-(lithocholoyl))-GLP-1(7-39);Gly⁸Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-39);Gly⁸Lys^(26,34)(N^(ε)-(lithocholoyl))-GLP-1(7-39);Arg²⁶Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-39);Gly⁸Arg²⁶Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-40);Lys²⁶(N^(ε)-(choloyl))Arg³⁴-GLP-1(7-40);Gly⁸Arg²⁶(N^(ε)-(choloyl))Arg³⁴-GLP-1(7-40);Arg^(26,34)Lys³⁶(N^(ε)-(choloyl))-GLP-1(7-40);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-(choloyl))-GLP-1(7-40);Lys²⁶(N^(ε)-(lithocholoyl))-GLP-1(7-40);Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-40);Lys^(26,34)-bis(N^(ε)-(lithocholoyl))-GLP-1(7-40);Gly⁸Lys²⁶(N^(ε)-(lithocholoyl))-GLP-1(7-40);Gly⁸Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-40);Gly⁸Lys^(26,34)-bis(N^(ε)-(lithocholoyl))-GLP-1(7-40);Arg²⁶Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-37);Lys²⁶(N^(ε)-(lithocholoyl))-GLP-1(7-36);Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-36);Lys^(26,34)-bis(N^(ε)-(lithocholoyl))-GLP-1(7-36);Gly⁸Lys²⁶(N^(ε)-(lithocholoyl))-GLP-1(7-36);Gly⁸Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-36);Gly⁸Lys^(26,34)-bis(N^(ε)-(lithocholoyl))-GLP-1(7-36);Arg²⁶Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-36);Lys²⁶(N^(ε)-(lithocholoyl))-GLP-1(7-35);Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-35);Lys^(26,34)-bis(N^(ε)-(lithocholoyl))-GLP-1(7-35);Gly⁸Lys²⁶(N^(ε)-(lithocholoyl))-GLP-1(7-35);Gly⁸Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-35);Gly⁸Lys^(26,34)-bis(N^(ε)-(lithocholoyl))-GLP-1(7-35);Arg²⁶Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-35);Lys²⁶(N^(ε)-(lithocholoyl))-GLP-1(7-36)amide;Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-36)amide;Lys^(26,34)-bis(N^(ε)-(lithocholoyl))-GLP-1(7-36)amide;Gly⁸Lys²⁶(N^(ε)-(lithocholoyl))-GLP-1(7-36)amide;Gly⁸Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-36)amide;Gly⁸Lys^(26,34)-bis(N^(ε)-(lithocholoyl))-GLP-1(7-36)amide;Arg²⁶Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-36)amide;Gly⁸Arg²⁶Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-37);Lys²⁶(N^(ε)-(lithocholoyl))Arg³⁴-GLP-1(7-37);Gly⁸Lys²⁶(N^(ε)-(lithocholoyl))Arg³⁴-GLP-1(7-37);Arg^(26,34)Lys³⁶(N^(ε)-(lithocholoyl))-GLP-1(7-37);Arg^(26,34)Lys³⁸(N^(ε)-(lithocholoyl))-GLP-1(7-37);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-(lithocholoyl))-GLP-1(7-37);Gly⁸Arg²⁶Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-38);Lys²⁶(N^(ε)-(lithocholoyl))Arg³⁴-GLP-1(7-38);Gly⁸Lys²⁶(N^(ε)-(lithocholoyl))Arg³⁴-GLP-1(7-38);Arg^(26,34)Lys³⁶(N^(ε)-(lithocholoyl))-GLP-1(7-38);Arg^(26,34)Lys³⁸(N^(ε)-(lithocholoyl))-GLP-1(7-38);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-(lithocholoyl))-GLP-1(7-38);Gly⁸Arg²⁶Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-39);Lys²⁶(N^(ε)-(lithocholoyl))Arg³⁴-GLP-1(7-39);Gly⁸Lys²⁶(N^(ε)-(lithocholoyl))Arg³⁴-GLP-1(7-39);Arg^(26,34)Lys³⁶(N^(ε)-(lithocholoyl))-GLP-1(7-39);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-(lithocholoyl))-GLP-1(7-39);Gly⁸Arg²⁶Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-40);Lys²⁶(N^(ε)-(lithocholoyl)Arg³⁴-GLP-1(7-40);Gly⁸Lys²⁶(N^(ε)-(lithocholoyl))Arg³⁴-GLP-1(7-40);Arg^(26,34)Lys³⁶(N^(ε)-(lithocholoyl))-GLP-1(7-40) andGly⁸Arg^(26,34)Lys³⁶(N^(ε)-(lithocholoyl))-GLP-1(7-40).

In a further preferred embodiment, the present invention relates to apharmaceutical composition comprising a GLP-1 derivative and apharmaceutically acceptable vehicle or carrier.

In a further preferred embodiment, the present invention relates to theuse of a GLP-1 derivative according to the invention for the preparationof a medicament which has a protracted profile of action relative toGLP-1(7-37).

In a further preferred embodiment, the present invention relates to theuse of a GLP-1 derivative according to the invention for the preparationof a medicament with protracted effect for the treatment of non-insulindependent diabetes mellitus.

In a further preferred embodiment, the present invention relates to theuse of a GLP-1 derivative according to the invention for the preparationof a medicament with protracted effect for the treatment of insulindependent diabetes mellitus.

In a further preferred embodiment, the present invention relates to theuse of a GLP-1 derivative according to the invention for the preparationof a medicament with protracted effect for the treatment of obesity.

In a further preferred embodiment, the present invention relates to amethod of treating insulin dependent or non-insulin dependent diabetesmellitus in a patient in need of such a treatment, comprisingadministering to the patient a therapeutically effective amount of aGLP-1 derivative of the invention, in particular a derivative ofGLP-1(7-C), wherein C is 35 or 36, together with a pharmaceuticallyacceptable carrier.

According to U.S. Pat. No. 5,631,224 (Novo Nordisk A/S) a strongsynergistic effect is observed in NIDDM patients by the combinedtreatment with GLP-1(7-37) or GLP-1(7-36)amide and an oral hypoglycemicagent.

Since pharmacodynamic and pharmacokinetic properties can be changedaccording to patients' demand by selecting a GLP-1 derivative of thepresent invention, additional therapeutic advantages can be gained bytreating the NIDDM patients in a regimen which additionally comprisestreatment with another antidiabetic agent.

Thus, the invention furthermore relates to the use of a GLP-1 derivativeaccording to the pre-sent invention for the preparation of a medicamentfor use in the treatment of diabetes in a regimen which additionallycomprises treatment with another antidiabetic agent.

In the present context the expression “antidiabetic agent” includescompounds for the treatment and/or prophylaxis of insulin resistance anddiseases wherein insulin resistance is the pathophysiological mechanism.

In one embodiment of this invention, the antidiabetic agent is insulinor an analogue an a derivative thereof.

In another embodiment the antidiabetic agent is a hypoglycaemic agent,preferably an oral hypoglycaemic agent.

Oral hypoglycaemic agents are preferably selected from the groupconsisting of sulfonylureas, biguanides, thiazolidinediones, glucosidaseinhibitors, glucagon antagonists, GLP-1 agonists, potassium channelopeners, insulin sensitizers, hepatic enzyme inhibitors, glucose uptakemodulators, compounds modifying the lipid metabolism, compounds loweringfood intake, and agents acting on the ATP-dependent potassium channel ofthe β-cells.

Among the sulfonylureas, tolbutamide, glibenclamide, glipizide andgliclazide are preferred.

Among the biguanides, metformin is preferred.

Among the thiazolidinediones, troglitazone and ciglitazone arepreferred.

Among the glucosidase inhibitors, acarbose is preferred.

Among the agents acting on the ATP-dependent potassium channel of theβ-cells the following are preferred: glibenclamide, glipizide,gliclazide, repaglinide.

U.S. Pat. No. 5,424,286 describes a method for stimulating insulinrelease with exendin polypeptide(s). The exendin polypeptides disclosedinclude HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGX; wherein X=P or Y, andHX1X2GTFITSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS; wherein X1X2=SD (exendin-3)or GE (exendin-4)). According to this document the insulinotropic effectof these polypeptides is greater than that attainable by administrationof GLP-1.

The exendin-3 and -4 and fragments are useful in treatment of diabetesmellitus (types I or II) and prevention of hyperglycaemia. Theynormalise hyperglycaemia through glucose-dependent, insulin-independentand insulin-dependent mechanisms. These insulinotropic peptides are moreactive than GLP-1. Exendin-4 is specific for exendin receptors, i.e. itdoes not interact with vasoactive intestinal peptide receptors.

WO 9746584 describes truncated versions of exendin peptide(s) fortreating diabetes. The disclosed peptides increase secretion andbiosynthesis of insulin, but reduce those of glucagon. The truncatedpeptides can be made more economically than full length versions.Compared with GLP-1 and the known exendins, they are more active(effective at lower doses), more stable to degradation and metabolismand have a longer lasting effect.

However, the high clearance limits the usefulness of these compounds,and thus there still is a need for improvements in this field.Accordingly, it is one object of the present invention to providederivatives of exendin and analogues thereof which have a protractedprofile of action relative to native exendin.

Thus, in one aspect the invention relates to an exendin derivativewherein at least one amino acid residue of the parent peptide has alipophilic substituent attached.

In a preferred embodiment only one lipophilic substituent is present.

In another preferred embodiment, the lipophilic substituent is attachedto the N-terminal amino acid residue.

In another preferred embodiment, the lipophilic substituent is attachedto the C-terminal amino acid residue.

In another preferred embodiment, the lipophilic substituent is attachedto an amino acid residue which is not the N-terminal or C-terminal aminoacid residue.

In further preferred embodiment, two lipophilic substituents arepresent.

In another preferred embodiment, one of the lipophilic substituents isattached to the N-terminal amino acid residue while the other isattached to the C-terminal amino acid residue.

In another preferred embodiment, one of the lipophilic substituents isattached to the C-terminal amino acid residue while the other isattached to an amino acid residue which is not the N-terminal orC-terminal amino acid residue.

In another preferred embodiment, both lipophilic substituents areattached to amino acid residues which are neither the N-terminal nor theC-terminal amino acid residue.

In further preferred embodiment, the lipophilic substituent comprisesfrom 4 to 40 carbon atoms, more preferred from 8 to 25 carbon atoms,such as 12 to 18 carbon atoms.

In another preferred embodiment, a lipophilic substituent is attached toan amino acid residue in such a way that a carboxyl group of thelipophilic substituent forms an amide bond with an amino group of theamino acid residue.

In another preferred embodiment, a lipophilic substituent is attached toan amino acid residue in such a way that an amino group of thelipophilic substituent forms an amide bond with a carboxyl group of theamino acid residue.

In another preferred embodiment, the lipophilic substituent is attachedto the parent peptide by means of a spacer.

In another preferred embodiment, the spacer is an unbranched alkaneα,ω-dicarboxylic acid group having from 1 to 7 methylene groups,preferably two methylene groups, which form a bridge between an aminogroup of the parent peptide and an amino group of the lipophilicsubstituent.

In another preferred embodiment, the spacer is an amino acid residueexcept cys, or a dipeptide such as gly-lys.

In another preferred embodiment, a carboxyl group of the parent peptideforms an amide bond with an amino group of lys or a dipeptide containinga lys residue, and the other amino group of the lys spacer or adipeptide spacer containing a lys residue forms an amide bond with acarboxyl group of the lipophilic substituent.

In another preferred embodiment, an amino group of the parent peptideforms an amide bond with a carboxylic group of the amino acid residue ordipeptide spacer, and an amino group of the amino acid residue ordipeptide spacer forms an amide bond with a carboxyl group of thelipophilic substituent.

In another preferred embodiment, a carboxyl group of the parent peptideforms an amide bond with an amino group of the amino acid residue spaceror dipeptide spacer, and a carboxyl group of the amino acid residuespacer or dipeptide spacer forms an amide bond with an amino group ofthe lipophilic substituent.

In another preferred embodiment, a carboxyl group of the parent peptideforms an amide bond with an amino group of a spacer which is asp or glu,or a dipeptide spacer containing an asp or glu residue, and a carboxylgroup of the spacer forms an amide bond with an amino group of thelipophilic substituent.

In one embodiment said spacer is γ-aminobutyroyl.

In a further preferred embodiment, the lipophilic substituent comprisesa partially or completely hydrogenated cyclopentanophenathrene skeleton.

In another preferred embodiment, the lipophilic substituent is anstraight-chain or branched alkyl group.

In another preferred embodiment, the lipophilic substituent is the acylgroup of a straight-chain or branched fatty acid.

In another preferred embodiment, the acyl group is selected from thegroup comprising CH₃(CH₂)_(n)CO—, wherein n is 4 to 38, preferablyCH₃(CH₂)₆CO—, CH₃(CH₂)₈CO—, CH₃(CH₂)₁₀CO—, CH₃(CH₂)₁₂CO—, CH₃(CH₂)₁₄CO—,CH₃(CH₂)₁₆CO—, CH₃(CH₂)₁₈CO—, CH₃(CH₂)₂₀CO— and CH₃(CH₂)₂₂CO—, mostpreferably hexadecanoyl.

In another preferred embodiment, the lipophilic substituent is an acylgroup of a straight-chain or branched alkane α,ω-dicarboxylic acid.

In another preferred embodiment, the acyl group is selected from thegroup comprising HOOC(CH₂)_(m)CO—, wherein m is from 4 to 38, preferablyfrom 4 to 24, more preferred selected from the group comprisingHOOC(CH₂)₁₄CO—, HOOC(CH₂)₁₆CO—, HOOC(CH₂)₁₈CO—, HOOC(CH₂)₂₀CO— andHOOC(CH₂)₂₂CO—.

In another preferred embodiment, the lipophilic substituent is a groupof the formula CH₃(CH₂)_(p)((CH₂)_(q)COOH)CHNH—CO(CH₂)₂CO—, wherein pand q are integers and p+q is an integer of from 8 to 33, preferablyfrom 12 to 28.

In another preferred embodiment, the lipophilic substituent is a groupof the formula CH₃(CH₂)_(r)CO—NHCH(COOH)(CH₂)₂CO—, wherein r is aninteger of from 10 to 24.

In another preferred embodiment, the lipophilic substituent is a groupof the formula CH₃(CH₂)_(s)CO—NHCH((CH₂)₂COOH)CO—, wherein s is aninteger of from 8 to 24.

In another preferred embodiment, the lipophilic substituent is a groupof the formula —NHCH(COOH)(CH₂)₄NH—CO(CH₂)_(u)CH₃, wherein u is aninteger of from 8 to 18.

In another preferred embodiment, the lipophilic substituent is a groupof the formula —NHCH(COOH)(CH₂)₄NH—COCH((CH₂)₂COOH)NH—CO(CH₂)_(w)CH₃,wherein w is an integer of from 10 to 16.

In another preferred embodiment, the lipophilic substituent is a groupof the formula —NHCH(COOH)(CH₂)₄NH—CO(CH₂)₂CH(COOH)NH—CO(CH₂)_(x)CH₃,wherein x is an integer of from 10 to 16.

In another preferred embodiment, the lipophilic substituent is a groupof the formula —NHCH(COOH)(CH₂)₄NH—CO(CH₂)₂CH(COOH)NH—CO(CH₂)_(y)CH₃,wherein y is zero or an integer of from 1 to 22.

In another preferred embodiment, the designation analogue comprisesderivatives wherein a total of up to fifteen, preferably up to ten aminoacid residues have been exchanged with any α-amino acid residue.

In another preferred embodiment, the designation analogue comprisesderivatives wherein a total of up to fifteen, preferably up to ten aminoacid residues have been exchanged with any α-amino acid residue whichcan be coded for by the genetic code.

In another preferred embodiment, the designation analogue comprisesderivatives wherein a total of up to six amino acid residues have beenexchanged with any α-amino acid residue which can be coded for by thegenetic code.

In another preferred embodiment, the parent peptide isHGEGTFTSDLSKQMEEEAVRLFIEWLKNGGX, wherein X=P or Y, or a fragment or ananalogue thereof.

In another preferred embodiment, the parent peptide isHX1X2GTFITSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS, wherein X1X2=SD or GE, or afragment or an analogue thereof.

In another preferred embodiment, the parent peptide isDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS, or a fragment or an analogue thereof.

In another preferred embodiment the exendin derivative is selected from

Arg¹⁸, Leu²⁰, Gln³⁴, Lys³³ (N^(ε)-(γ-aminobutyroyl(N^(α)-hexadecanoyl)))Exendin-4-(7-45)-NH₂, Arg³³, Leu²⁰, Gln³⁴, Lys¹⁸(N^(ε)-(γ-aminobutyroyl(N^(α)-hexadecanoyl))) Exendin-4-(7-45)-NH₂.

The present invention furthermore relates to a pharmaceuticalcomposition comprising an exendin derivative according to the presentinvention and a pharmaceutically acceptable vehicle or carrier.

Moreover, the invention is concerned with the use of an exendinderivative according to the present invention for the preparation of amedicament which has a protracted profile of action relative to exendin.

The invention also relates to the use of an exendin derivative accordingto the present invention for the preparation of a medicament with aprotracted profile of action for the treatment of non-insulin dependentdiabetes mellitus or for the treatment of insulin dependent diabetesmellitus or for the treatment of obesity.

The invention also relates to a method of treating insulin dependent ornon-insulin dependent diabetes mellitus in a patient in need of such atreatment, comprising administering to the patient a therapeuticallyeffective amount of a exendin derivative according to the presentinvention together with a pharmaceutically acceptable carrier.

DETAILED DESCRIPTION OF THE INVENTION

To obtain a satisfactory protracted profile of action of the GLP-1derivative, the lipophilic substituent attached to the GLP-1 moietypreferably comprises 4-40 carbon atoms, in particular 8-25 carbon atoms.The lipophilic substituent may be attached to an amino group of theGLP-1 moiety by means of a carboxyl group of the lipophilic substituentwhich forms an amide bond with an amino group of the amino acid residueto which it is attached. Alternatively, the lipophilic substituent maybe attached to said amino acid residue in such a way that an amino groupof the lipophilic substituent forms an amide bond with a carboxyl groupof the amino acid residue. As a further option, the lipophilicsubstituent may be linked to the GLP-1 moiety via an ester bond.Formally, the ester can be formed either by reaction between a carboxylgroup of the GLP-1 moiety and a hydroxyl group of the substituent-to-beor by reaction between a hydroxyl group of the GLP-1 moiety and acarboxyl group of the substituent-to-be. As a further alternative, thelipophilic substituent can be an alkyl group which is introduced into aprimary amino group of the GLP-1 moiety.

In one preferred embodiment of the invention, the lipophilic substituentis attached to the GLP-1 moiety by means of a spacer in such a way thata carboxyl group of the spacer forms an amide bond with an amino groupof the GLP-1 moiety. Examples of suitable spacers are succinic acid,Lys, Glu or Asp, or a dipeptide such as Gly-Lys. When the spacer issuccinic acid, one carboxyl group thereof may form an amide bond with anamino group of the amino acid residue, and the other carboxyl groupthereof may form an amide bond with an amino group of the lipophilicsubstituent. When the spacer is Lys, Glu or Asp, the carboxyl groupthereof may form an amide bond with an amino group of the amino acidresidue, and the amino group thereof may form an amide bond with acarboxyl group of the lipophilic substituent. When Lys is used as thespacer, a further spacer may in some instances be inserted between theε-amino group of Lys and the lipophilic substituent. In one preferredembodiment, such a further spacer is succinic acid which forms an amidebond with the ε-amino group of Lys and with an amino group present inthe lipophilic substituent. In another preferred embodiment such afurther spacer is Glu or Asp which forms an amide bond with the ε-aminogroup of Lys and another amide bond with a carboxyl group present in thelipophilic substituent, that is, the lipophilic substituent is aN^(ε)-acylated lysine residue.

In another preferred embodiment of the present invention, the lipophilicsubstituent has a group which can be negatively charged. One preferredgroup which can be negatively charged is a carboxylic acid group.

The parent peptide can be produced by a method which comprises culturinga host cell containing a DNA sequence encoding the polypeptide andcapable of expressing the polypeptide in a suitable nutrient mediumunder conditions permitting the expression of the peptide, after whichthe resulting peptide is recovered from the culture.

The medium used to culture the cells may be any conventional mediumsuitable for growing the host cells, such as minimal or complex mediacontaining appropriate supplements. Suitable media are available fromcommercial suppliers or may be prepared according to published recipes(e.g. in catalogues of the American Type Culture Collection). Thepeptide produced by the cells may then be recovered from the culturemedium by conventional procedures including separating the host cellsfrom the medium by centrifugation or filtration, precipitating theproteinaceous components of the supernatant or filtrate by means of asalt, e.g. ammonium sulphate, purification by a variety ofchromatographic procedures, e.g. ion exchange chromatography, gelfiltration chromatography, affinity chromatography, or the like,dependent on the type of peptide in question.

The DNA sequence encoding the parent peptide may suitably be of genomicor cDNA origin, for instance obtained by preparing a genomic or cDNAlibrary and screening for DNA sequences coding for all or part of thepeptide by hybridisation using synthetic oligonucleotide probes inaccordance with standard techniques (see, for example, Sambrook, J,Fritsch, E F and Maniatis, T, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, New York, 1989). The DNA sequenceencoding the peptide may also be prepared synthetically by establishedstandard methods, e.g. the phosphoamidite method described by Beaucageand Caruthers, Tetrahedron Letters 22 (1981), 1859-1869, or the methoddescribed by Matthes et al., EMBO Journal 3 (1984), 801-805. The DNAsequence may also be prepared by polymerase chain reaction usingspecific primers, for instance as described in U.S. Pat. No. 4,683,202or Saiki et al., Science 239 (1988), 487-491.

The DNA sequence may be inserted into any vector which may convenientlybe subjected to recombinant DNA procedures, and the choice of vectorwill often depend on the host cell into which it is to be introduced.Thus, the vector may be an autonomously replicating vector, i.e. avector which exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g. a plasmid.Alternatively, the vector may be one which, when introduced into a hostcell, is integrated into the host cell genome and replicated togetherwith the chromosome(s) into which it has been integrated.

The vector is preferably an expression vector in which the DNA sequenceencoding the peptide is operably linked to additional segments requiredfor transcription of the DNA, such as a promoter. The promoter may beany DNA sequence which shows transcriptional activity in the host cellof choice and may be derived from genes encoding proteins eitherhomologous or heterologous to the host cell. Examples of suitablepromoters for directing the transcription of the DNA encoding thepeptide of the invention in a variety of host cells are well known inthe art, cf. for instance Sambrook et al., supra.

The DNA sequence encoding the peptide may also, if necessary, beoperably connected to a suitable terminator, polyadenylation signals,transcriptional enhancer sequences, and translational enhancersequences. The recombinant vector of the invention may further comprisea DNA sequence enabling the vector to replicate in the host cell inquestion.

The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell or one whichconfers resistance to a drug, e.g. ampicillin, kanamycin, tetracyclin,chloramphenicol, neomycin, hygromycin or methotrexate.

To direct a parent peptide of the present invention into the secretorypathway of the host cells, a secretory signal sequence (also known as aleader sequence, prepro sequence or pre sequence) may be provided in therecombinant vector. The secretory signal sequence is joined to the DNAsequence encoding the peptide in the correct reading frame. Secretorysignal sequences are commonly positioned 5′ to the DNA sequence encodingthe peptide. The secretory signal sequence may be that normallyassociated with the peptide or may be from a gene encoding anothersecreted protein.

The procedures used to ligate the DNA sequences coding for the presentpeptide, the promoter and optionally the terminator and/or secretorysignal sequence, respectively, and to insert them into suitable vectorscontaining the information necessary for replication, are well known topersons skilled in the art (cf., for instance, Sambrook et al., supra).

The host cell into which the DNA sequence or the recombinant vector isintroduced may be any cell which is capable of producing the presentpeptide and includes bacteria, yeast, fungi and higher eukaryotic cells.Examples of suitable host cells well known and used in the art are,without limitation, E. coli, Saccharomyces cerevisiae, or mammalian BHKor CHO cell lines.

Examples of compounds which can be useful as GLP-1 moieties according tothe present invention are described in International Patent ApplicationNo. WO 87/06941 (The General Hospital Corporation) which relates to apeptide fragment which comprises GLP-1(7-37) and functional derivativesthereof and to its use as an insulinotropic agent.

Further GLP-1 analogues are described in International PatentApplication No. 90/11296 (The General Hospital Corporation) whichrelates to peptide fragments which comprise GLP-1(7-36) and functionalderivatives thereof and have an insulinotropic activity which exceedsthe insulinotropic activity of GLP-1(1-36) or GLP-1(1-37) and to theiruse as insulinotropic agents.

International Patent Application No. 91/11457 (Buckley et al.) disclosesanalogues of the active GLP-1 peptides 7-34, 7-35, 7-36, and 7-37 whichcan also be useful as GLP-1 moieties according to the present invention.

Pharmaceutical Compositions

Pharmaceutical compositions containing a GLP-1 derivative according tothe present invention may be administered parenterally to patients inneed of such a treatment. Parenteral administration may be performed bysubcutaneous, intramuscular or intravenous injection by means of asyringe, optionally a pen-like syringe. Alternatively, parenteraladministration can be performed by means of an infusion pump. A furtheroption is a composition which may be a powder or a liquid for theadministration of the GLP-1 derivative in the form of a nasal orpulmonal spray. As a still further option, the GLP-1 derivatives of theinvention can also be administered transdermally, e.g. from a patch,optionally a iontophoretic patch, or transmucosally, e.g. bucally.

Pharmaceutical compositions containing a GLP-1 derivative of the presentinvention may be prepared by conventional techniques, e.g. as describedin Remington's Pharmaceutical Sciences, 1985 or in Remington: TheScience and Practice of Pharmacy, 19^(th) edition, 1995.

Thus, the injectable compositions of the GLP-1 derivative of theinvention can be prepared using the conventional techniques of thepharmaceutical industry which involves dissolving and mixing theingredients as appropriate to give the desired end product.

According to one procedure, the GLP-1 derivative is dissolved in anamount of water which is somewhat less than the final volume of thecomposition to be prepared. An isotonic agent, a preservative and abuffer is added as required and the pH value of the solution isadjusted—if necessary—using an acid, e.g. hydrochloric acid, or a base,e.g. aqueous sodium hydroxide as needed. Finally, the volume of thesolution is adjusted with water to give the desired concentration of theingredients.

Examples of isotonic agents are sodium chloride, mannitol and glycerol.

Examples of preservatives are phenol, m-cresol, methyl p-hydroxybenzoateand benzyl alcohol.

Examples of suitable buffers are sodium acetate and sodium phosphate.

Further to the above-mentioned components, solutions containing a GLP-1derivative according to the present invention may also contain asurfactant in order to improve the solubility and/or the stability ofthe GLP-1 derivative.

A composition for nasal administration of certain peptides may, forexample, be prepared as described in European Patent No. 272097 (to NovoNordisk A/S) or in WO 93/18785.

According to one preferred embodiment of the present invention, theGLP-1 derivative is provided in the form of a composition suitable foradministration by injection. Such a composition can either be aninjectable solution ready for use or it can be an amount of a solidcomposition, e.g. a lyophilised product, which has to be dissolved in asolvent before it can be injected. The injectable solution preferablycontains not less than about 2 mg/ml, preferably not less than about 5mg/ml, more preferred not less than about 10 mg/ml of the GLP-1derivative and, preferably, not more than about 100 mg/ml of the GLP-1derivative.

The GLP-1 derivatives of this invention can be used in the treatment ofvarious diseases. The particular GLP-1 derivative to be used and theoptimal dose level for any patient will depend on the disease to betreated and on a variety of factors including the efficacy of thespecific peptide derivative employed, the age, body weight, physicalactivity, and diet of the patient, on a possible combination with otherdrugs, and on the severity of the case. It is recommended that thedosage of the GLP-1 derivative of this invention be determined for eachindividual patient by those skilled in the art.

In particular, it is envisaged that the GLP-1 derivative will be usefulfor the preparation of a medicament with a protracted profile of actionfor the treatment of non-insulin dependent diabetes mellitus and/or forthe treatment of obesity.

The present invention is further illustrated by the following exampleswhich, however, are not to be construed as limiting the scope ofprotection. The features disclosed in the foregoing description and inthe following examples may, both separately and in any combinationthereof, be material for realising the invention in diverse formsthereof.

EXAMPLES

The following acronyms for commercially available chemicals are used:

DMF: N,N-Dimethylformamide. DCC: N,N-Dicyclohexylcarbodiimide NMP:N-Methyl-2-pyrrolidone. EDPA: N-Ethyl-N,N-diisopropylamine. EGTA:Ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′- tetraacetic acid. GTPGuanosine 5′-triphosphate. TFA: Trifluoroacetic acid. THF:Tetrahydrofuran H-Glu(OH)-OBu^(t): L-Glutamic acid α-tert-butyl esterCap-ONSu: Octanoic acid 2,5-dioxopyrrolidin-1-yl ester Lau-ONSu:Dodecanoic acid 2,5-dioxopyrrolidin-1-yl ester Myr-ONSu: Tetradecanoicacid 2,5-dioxopyrrolidin-1-yl ester. Pal-ONSu: Hexadecanoic acid2,5-dioxopyrrolidin-1-yl ester. Ste-ONSu Octadecanoic acid2,5-dioxopyrrolidin-1-yl ester.

Abbreviations:

-   PDMS: Plasma Desorption Mass Spectrometry-   MALDI-MS: Matrix Assisted Laser Desorption/Ionisation Mass    Spectrometry-   HPLC: High Performance Liquid Chromatography-   amu: atomic mass units-   Lit-Glu(ONSu)-OBu^(t)N^(α)-Lithochoyl-L-glutamic acid α-t-butyl    ester γ-2,5-dioxopyrrolidin-1-yl ester-   Cap-Glu(ONSu)-OBu^(t): N^(α)-Octanoyl-L-glutamic acid α-t-butyl    ester γ-2,5-dioxopyrrolidin-1-yl ester-   Cac-Glu(ONSu)-OBu^(t): N^(α)-Decanoyl-L-glutamic acid α-t-butyl    ester γ-2,5-dioxopyrrolidin-1-yl ester-   Lau-Glu(ONSu)-OBu^(t): N^(α)-Dodecanoyl-L-glutamic acid α-t-butyl    ester γ-2,5-dioxopyrrolidin-1-yl ester-   Myr-Glu(ONSu)-OBu^(t): N^(α)-Tetradecanoyl-L-glutamic acid α-t-butyl    ester γ-2,5-dioxopyrrolidin-1-yl ester-   Pal-Glu(ONSu)-OBu^(t): N^(α)-Hexadecanoyl-(L)-glutamic acid    α-t-butyl-γ-2,5-dioxopyrrolidin-1-yl diester.-   Ste-Glu(ONSu)-OBu^(t): N^(α)-Octadecanoyl-(L)-glutamic acid    α-t-butyl-γ-2,5-dioxopyrrolidin-1-yl diester-   Lau-β-Ala-ONSu: N^(β)-Dodecanoyl-β-alanine 2,5-dioxopyrrolidin-1-yl    ester-   Pal-β-Ala-ONSu: N^(β)-Hexadecanoyl-β-alanine    2,5-dioxopyrrolidin-1-yl ester-   Lau-GABA-ONSu: N^(γ)-Dodecanoyl-γ-aminobutyric acid    2,5-dioxopyrrolidin-1-yl ester-   Myr-GABA-ONSu: N^(γ)-Tetradecanoyl-γ-aminobutyric acid    2,5-dioxopyrrolidin-1-yl ester-   Pal-GABA-ONSu: N^(γ)-Hexadecanoyl-γ-aminobutyric acid    2,5-dioxopyrrolidin-1-yl ester-   Ste-GABA-ONSu: N^(γ)-Octadecanoyl-γ-aminobutyric acid    2,5-dioxopyrrolidin-1-yl ester-   Pal-Isonip-ONSu: N-Hexadecanoyl-piperidine-4-carboxylic acid    2,5-dioxopyrrolidin-1-yl ester-   Pal-Glu(OBu^(t))-ONSu: N^(α)-Hexadecanoyl-L-glutamic acid    α-2,5-dioxopyrrolidin-1-yl ester γ-t-butyl ester-   HOOC—(CH₂)₆—COONSu: ω-Carboxyheptanoic acid 2,5-dioxopyrrolidin-1-yl    ester.-   HOOC—(CH₂)₁₀—COONSu: ω-Carboxyundecanoic acid    2,5-dioxopyrrolidin-1-yl ester.-   HOOC—(CH₂)₁₂—COONSu: ω-Carboxytridecanoic acid    2,5-dioxopyrrolidin-1-yl ester.-   HOOC—(CH₂)₁₄—COONSu: ω-Carboxypentadecanoic acid    2,5-dioxopyrrolidin-1-yl ester.-   HOOC—(CH₂)₁₆—COONSu: ω-Carboxyheptadecanoic acid    2,5-dioxopyrrolidin-1-yl ester.-   HOOC—(CH₂)₁₈—COONSu: ω-Carboxynonadecanoic acid    2,5-dioxopyrrolidin-1-yl ester.

Analytical Plasma Desorption Mass Spectrometry Sample Preparation:

The sample is dissolved in 0.1% TFA/EtOH (1:1) at a concentration of 1μg/μl. The sample solution (5-10 μg) is placed on a nitrocellulosetarget (Bio-ion AB, Uppsala, Sweden) and allowed to adsorb to the targetsurface for 2 minutes. The target is subsequently rinsed with 2×25 μl0.1% TFA and spin-dried. Finally, the nitrocellulose target is placed ina target carrousel and introduced into the mass spectrometer.

MS Analysis:

PDMS analysis was carried out using a Bio-ion 20 time-of flightinstrument (Bio-ion Nordic AB, Uppsala, Sweden). An acceleration voltageof 15 kV was applied and molecular ions formed by bombardment of thenitrocellulose surface with 252-Cf fission fragments were acceleratedtowards a stop detector. The resulting time-of-flight spectrum wascalibrated into a true mass spectrum using the H⁺ and NO⁺ ions at m/z 1and 30, respectively. Mass spectra were generally accumulated for1.0×10⁶ fission events corresponding to 15-20 minutes. Resultingassigned masses all correspond to isotopically averaged molecularmasses. The accuracy of mass assignment is generally better than 0.1%.

MALDI-MS

MALDI-TOF MS analysis was carried out using a Voyager RP instrument(PerSeptive Biosystems Inc., Framingham, Mass.) equipped with delayedextraction and operated in linear mode. Alpha-cyano-4-hydroxy-cinnamicacid was used as matrix, and mass assignments were based on externalcalibration.

Example 1 Synthesis of N^(α)-hexadecanoyl-Glu(ONSu)-OBu^(t)

To a suspension of H-Glu(OH)—OBu^(t) (4.2 g, 20.6 mmol), DMF (500 ml)and EDPA (2.65 g, 20.6 mmol) was added drop by drop a solution ofPal-ONSu (7.3 g, 20.6 mmol) in DMF (100 ml). The reaction mixture wasstirred for 64 h at room temperature and then concentrated in vacuo to atotal volume of 20 ml. The residue was partitioned between 10% aqueouscitric acid (300 ml) and ethyl acetate (250 ml), and the phases wereseparated. The organic phase was concentrated in vacuo and the residuedissolved in DMF (50 ml). The resulting solution was added drop by dropto a 10% aqueous solution of citric acid (500 ml) kept at 0° C. Theprecipitated compound was collected and washed with iced water and driedin a vacuum drying oven. The dried compound was dissolved in DMF (45 ml)and HONSu (2.15 g, 18.7 mmol) was added. To the resulting mixture wasadded a solution of N,N′-dicyclohexylcarbodiimide (3.5 g, 17 mmol) indichloromethane (67 ml). The reaction mixture was stirred for 16 h atroom temperature, and the precipitated compound was filtered off. Theprecipitate was recrystallised from n-heptane/2-propanol to give thetitle compound (6.6 g, 72%).

Example 2 Synthesis of N^(α)-octadecanoyl-Glu(ONSu)-OBu^(t)

To a suspension of H-Glu(OH)—OBu^(t)(2.82 g, 13.9 mmol), DMF (370 ml)and EDPA (1.79 g, 13.9 mmol) was added drop by drop a solution ofSte-ONSu (5.3 g, 13.9 mmol) in DMF (60 ml). Dichloromethane (35 ml) wasadded, and the reaction mixture was stirred for 24 h at room temperatureand then concentrated in vacuo. The residue was partitioned between 10%aqueous citric acid (330 ml) and ethyl acetate (200 ml), and the phaseswere separated. The organic phase was concentrated in vacuo and theresidue dissolved in DMF (60 ml). The resulting solution was added dropby drop to a 10% aqueous solution of citric acid (400 ml) kept at 0° C.The precipitated compound was collected and washed with iced water anddried in a vacuum drying oven. The dried compound was dissolved in DMF(40 ml) and HONSu (1.63 g, 14.2 mmol) was added. To the resultingmixture was added a solution of DCC (2.66 g, 12.9 mmol) indichloromethane (51 ml). The reaction mixture was stirred for 64 h atroom temperature, and the precipitated compound was filtered off. Theprecipitate was recrystallised from n-heptane/2-propanol to give thetitle compound (4.96 g, 68%).

Example 3 Synthesis of Arg^(26,34), Lys³⁶(N^(ε)-(γ-glutamyl(N^(α)-hexadecanoyl))) GLP-1 (7-36)-OH

To a mixture of Arg^(26,34), Lys³⁶ GLP-1 (7-36)-OH (12.2 mg, 3.67 μmol),EDPA (13.3 mg, 103 μmol), NMP (1.71 ml) and water (855 μl) was added asolution of Pal-Glu(ONSu)-OBu^(t) (5.94 mg, 11 μmol), prepared asdescribed above, in NMP (148 μl). The reaction mixture was gently shakenfor 5 min. at room temperature, and then allowed to stand for anadditional 90 min. at room temperature. The reaction was quenched by theaddition of a solution of glycine (6 mg, 81 μmol) in water (0.6 ml). A0.5% aqueous solution of ammonium-acetate (38 ml) was added, and theresulting mixture eluted onto a Varian 5 g C8 Mega Bond Elut®, theimmobilised compound washed with 5% aqueous acetonitril (20 ml), andfinally liberated from the cartridge by elution with TFA (25 ml). Theeluate was concentrated in vacuo, and the residue purified by columnchromatography using a cyanopropyl column (Zorbax 300SB-CN) and astandard acetonitril/TFA system. The column was heated to 65° C. and theacetonitril gradient was 0-100% in 60 minutes. The title compound (3.1mg, 23%) was isolated, and the product was analysed by PDMS. The m/zvalue for the protonated molecular ion was found to be 3695±3. Theresulting molecular weight is thus 3694±3 amu (theoretical value 3694amu).

Example 4 Synthesis of Arg^(26,34), Lys³⁶(N^(ε)-(γ-glutamyl(N-octadecanoyl))) GLP-1 (7-36)-OH

To a mixture of Arg^(26,34), Lys³⁶ GLP-1 (7-36)-OH (12.2 mg, 3.7 μmol),EDPA (13.3 mg, 103 μmol), NMP (1.71 ml) and water (855 μl) was added asolution of Ste-Glu(ONSu)-OBu^(t) (6.25 mg, 11 μmol), prepared as above,in NMP (1 ml). The reaction mixture was gently shaken for 5 min. at roomtemperature, and then allowed to stand for an additional 90 min. at roomtemperature. The reaction was quenched by the addition of a solution ofglycine (6 mg, 81 μmol) in water (0.6 ml). A 0.5% aqueous solution ofammonium acetate (54 ml) was added, and the resulting mixture elutedonto a Varian 5 g C8 Mega Bond Elut®, the immobilised compound washedwith 5% aqueous acetonitril (20 ml), and finally liberated from thecartridge by elution with TFA (25 ml). The eluate was concentrated invacuo, and the residue purified by column chromatography using acyanopropyl column (Zorbax 300SB-CN) and a standard acetonitril/TFAsystem. The column was heated to 65° C. and the acetonitril gradient was0-100% in 60 minutes. The title compound (3.7 mg, 27%) was isolated, andthe product was analysed by PDMS. The m/z value for the protonatedmolecular ion was found to be 3723±3. The resulting molecular weight isthus 3722±3 amu (theoretical value 3722 amu).

Example 5 Synthesis of Arg¹⁸, Leu²⁰, Gln³⁴, Lys³³(N^(ε)-(γ-aminobutyroyl(N^(α)-hexadecanoyl))) Exendin-4-(7-45)-NH₂

To a mixture of Arg¹⁸, Leu²⁰, Gln³⁴-Exendin-4-NH₂ (9.7 mg, 2.3 μmol),EDPA (8.4 mg, 64.7 μmol), NMP (1.36 ml) and water (0.68 ml) was added asolution of Pal-GABA-ONSu (3 mg, 6.9 μmol) in NMP (76 μl). The reactionmixture was gently shaken for 5 min., and then allowed to stand for anadditional 90 min. at room temperature. The reaction was quenched by theaddition of a solution of glycine (3.8 mg, 50.8 μmol) in water (38 μl).The resulting mixture was purified by column chromatography using acyanopropyl column (Zorbax 300SB-CN) and a standard acetonitril/TFAsystem. The column was heated to 65° C. and the acetonitril gradient was0-100% in 60 minutes. The title compound (4.5 mg, 43%) was isolated, andthe product was analysed by PDMS. The m/z value for the protonatedmolecular ion was found to be 4532.8±3. The resulting molecular weightis thus 4531.8±3 amu (theoretical value 4534 amu).

Example 6 Synthesis of Arg³³, Leu²⁰, Gln³⁴, Lys¹⁸(N^(ε)-(γ-aminobutyroyl(N-hexadecanoyl))) Exendin-4-(7-45)-NH₂

To a mixture of Arg³³, Leu²⁰, Gln³⁴-Exendin-4-NH₂ (10 mg, 2.4 μmol),EDPA (8.6 mg, 66.5 μmol), NMP (1.4 ml) and water (0.7 ml) was added asolution of Pal-GABA-ONSu (3.1 mg, 7.1 μmol) in NMP (78 μl). Thereaction mixture was gently shaken for 5 min., and then allowed to standfor an additional 145 min. at room temperature. The reaction wasquenched by the addition of a solution of glycine (3.9 mg, 52.3 μmol) inwater (39 μl). The resulting mixture was purified by columnchromatography using a cyanopropyl column (Zorbax 300SB-CN) and astandard acetonitril/TFA system. The column was heated to 65° C. and theacetonitril gradient was 0-100% in 60 minutes. The title compound (2.9mg, 21%) was isolated, and the product was analysed by PDMS. The m/zvalue for the protonated molecular ion was found to be 4533.8±3. Theresulting molecular weight is thus 4532.8±3 amu (theoretical value 4534amu).

Biological Findings

Protraction of GLP-1 Derivatives After s.c. Administration

The protraction of a number GLP-1 derivatives of the invention wasdetermined by monitoring the concentration thereof in plasma after scadministration to healthy pigs, using the method described below. Forcomparison also the concentration in plasma of GLP-1(7-37) after sc.administration was followed. The protraction of other GLP-1 derivativesof the invention can be determined in the same way.

Pigs (50% Duroc, 25% Yorkshire, 25% Danish Landrace, app 40 kg) werefasted from the beginning of the experiment. To each pig 0.5 nmol oftest compound per kg body weight was administered in a 50 mM isotonicsolution (5 mM phosphate, pH 7.4, 0.02% Tween®-20 (Merck), 45 mg/mlmannitol (pyrogen free, Novo Nordisk). Blood samples were drawn from acatheter in vena jugularis. 5 ml of the blood samples were poured intochilled glasses containing 175 μl of the following solution: 0.18 MEDTA, 1500 KIE/ml aprotinin (Novo Nordisk) and 3% bacitracin (Sigma), pH7.4. Within 30 min, the samples were centrifuged for 10 min at 5-6000*g.Temperature was kept at 4° C. The supernatant was pipetted intodifferent glasses and kept at minus 20° C. until use.

The plasma concentrations of the peptides were determined by RIA using amonoclonal antibody specific for the N-terminal region of GLP-1(7-37).The cross reactivities were less than 1% with GLP-1(1-37) andGLP-1(8-36)amide and <0.1% with GLP-1(9-37), GLP-1(10-36)amide andGLP-1(11-36)amide. The entire procedure was carried out at 4° C.

The assay was carried out as follows: 100 μl plasma was mixed with 271μl 96% ethanol, mixed using a vortex mixer and centrifuged at 2600*9 for30 min. The supernatant was decanted into Minisorp tubes and evaporatedcompletely (Savant Speedvac AS290). The evaporation residue wasreconstituted in the assay buffer consisting of 80 mM NaH₂PO₄/Na₂HPO₄,0.1% HSA (Orpha 20/21, Behring), 10 mM EDTA, 0.6 mM thiomersal (Sigma),pH 7.5. Samples were reconstituted in volumes suitable for theirexpected concentrations, and were allowed to reconstitute for 30 min. To300 μl sample, 100 μl antibody solution in dilution buffer containing 40mM NaH₂PO₄/Na₂HPO₄, 0.1% HSA, 0.6 mM thiomersal, pH 7.5, was added. Anon-specific sample was prepared by mixing 300 μl buffer with 100 μldilution buffer. Individual standards were prepared from freeze driedstocks, dissolved in 300 μl assay buffer. All samples were pre-incubatedin Minisorp tubes with antibody as described above for 72 h. 200 μltracer in dilution buffer containing 6-7000 CPM was added, samples weremixed and incubated for 48 h. 1.5 ml of a suspension of 200 ml per litreof heparin-stabilised bovine plasma and 18 g per litre of activatedcarbon (Merck) in 40 mM NaH₂PO₄/Na₂HPO₄, 0.6 mM thiomersal, pH 7.5, wasadded to each tube. Before use, the suspension was mixed and allowed tostand for 2 h at 4° C. All samples were incubated for 1 h at 4° C. andthen centrifuged at 3400*g for 25 min. Immediately after the 4centrifugation, the supernatant was decanted and counted in a γ-counter.The concentration in the samples was calculated from individual standardcurves. Plasma concentrations were found, calculated as % of the maximumconcentration for the individual compounds (n=2). The GLP-1 derivativesof the invention have a protracted profile of action relative toGLP-1(7-37) and are much more persistent in plasma than GLP-1(7-37). Thetime at which the peak concentration in plasma is achieved varies withinwide limits, depending on the particular GLP-1 derivative selected.

Stimulation of cAMP Formation in a Cell Line Expressing the Cloned HumanGLP-1 Receptor

In order to demonstrate efficacy of the GLP-1 derivatives, their abilityto stimulate formation of cAMP in a cell line expressing the clonedhuman GLP-1 receptor was tested. An EC₅₀ was calculated from thedose-response curve.

Baby hamster kidney (BHK) cells expressing the human pancreatic GLP-1receptor were used (Knudsen and Pridal, 1996, Eur. J. Pharm. 318,429-435). Plasma membranes were prepared (Adelhorst et al, 1994, J.Biol. Chem. 269, 6275) by homogenisation in buffer (10 mmol/l Tris-HCland 30 mmol/l NaCl pH 7.4, containing, in addition, 1 mmol/ldithiothreitol, 5 mg/l leupeptin (Sigma, St. Louis, Mo., USA), 5 mg/lpepstatin (Sigma, St. Louis, Mo., USA), 100 mg/l bacitracin (Sigma, St.Louis, Mo., USA), and 16 mg/l aprotinin (Novo Nordisk A/S, Bagsvaerd,Denmark)). The homogenate was centrifuged on top of a layer of 41 w/v %sucrose. The white band between the two layers was diluted in buffer andcentrifuged. Plasma membranes were stored at −80° C. until used.

The assay was carried out in 96-well microtiter plates in a total volumeof 140 μl. The buffer used was 50 mmol/l Tris-HCl, pH 7.4 with theaddition of 1 mmol/l EGTA, 1.5 mmol/l MgSO₄, 1.7 mmol/l ATP, 20 mM GTP,2 mmol/l 3-isobutyl-1-methylxanthine, 0.01% Tween-20 and 0.1% humanserum albumin (Reinst, Behringwerke AG, Marburg, Germany). Compounds tobe tested for agonist activity were dissolved and diluted in buffer,added to the membrane preparation and the mixture was incubated for 2 hat 37° C. The reaction was stopped by the addition of 25 μl of 0.05mol/l HCl. Samples were diluted 10 fold before analysis for cAMP by ascintillation proximity assay (RPA 538, Amersham, UK).

1-91. (canceled)
 92. A derivative of an analogue of exendin-4 where saidanalogue has an amino acid sequence that differs from the amino acidsequence of exendin-4 by the substitution of up to ten amino acidresidues with any α-amino acid residue, and wherein said derivative hasone lipophilic substituent attached, optionally via a spacer, to anamino acid residue of said analogue which is not the N-terminal orC-terminal amino acid residue of said analogue.
 93. The derivative ofclaim 92, wherein said analogue has an amino acid sequence that differsfrom the amino acid sequence of exendin-4 by the substitution of up tosix amino acid residues with any α-amino acid residue
 94. The derivativeof claim 92, wherein the lipophilic substituent has 4 to 40 carbonatoms.
 95. The derivative of claim 94, wherein the lipophilicsubstituent has 8 to 25 carbon atoms.
 96. The derivative of claim 94,wherein the lipophilic substituent is attached by means of a spacer. 97.The derivative of claim 96, wherein the spacer is an unbranched alkaneα,ω-dicarboxylic acid group having from 1 to 7 methylene groups.
 98. Thederivative of claim 97, wherein the spacer is an unbranched alkaneα,ω-dicarboxylic acid group having two methylene groups.
 99. Thederivative of claim 96, wherein the spacer is an amino acid residueexcept cys, or a dipeptide such as gly-lys.
 100. The derivative of claim94, wherein the lipophilic substituent is a partially or completelyhydrogenated cyclopentanophenathrene skeleton.
 101. The derivative ofclaim 94, wherein the lipophilic substituent is a straight-chain orbranched alkyl group.
 102. The derivative of claim 94, wherein thelipophilic substituent is a straight-chain or branched acyl group. 103.The derivative of claim 102, wherein the acyl group is of the formulaCH₃(CH₂)_(n)CO—, wherein n is 4 to
 38. 104. The derivative of claim 103,wherein the acyl group is CH₃(CH₂)₆CO—, CH₃(CH₂)₈CO—, CH₃(CH₂)₁₀CO—,CH₃(CH₂)₁₂CO—, CH₃(CH₂)₁₄CO—, CH₃(CH₂)₁₆CO—, CH₃(CH₂)₁₈CO—,CH₃(CH₂)₂₀CO— or CH₃(CH₂)₂₂CO—.
 105. The derivative of claim 94, whereinthe lipophilic substituent is an acyl group of a straight-chain orbranched alkane α,ω-dicarboxylic acid.
 106. The derivative of claim 105,wherein the acyl group is of the formula HOOC(CH₂)_(m)CO—, wherein m isfrom 4 to
 38. 107. The derivative of claim 106, wherein the acyl groupis HOOC(CH₂)₁₄CO—, HOOC(CH₂)₁₆CO—, HOOC(CH₂)₁₈CO—, HOOC(CH₂)₂₀CO— orHOOC(CH₂)₂₂CO—.
 108. The derivative of claim 94, wherein the lipophilicsubstituent is a group of the formulaCH₃(CH₂)_(p)((CH₂)_(q)COOH)CHNH—CO(CH₂)₂CO—, wherein p and q areintegers and p+q is an integer of from 8 to
 33. 109. The derivative ofclaim 94, wherein the lipophilic substituent is a group of the formulaCH₃(CH₂)_(r)CO—NHCH(COOH)(CH₂)₂CO—, wherein r is an integer of from 10to
 24. 110. The derivative of claim 94, wherein the lipophilicsubstituent is a group of the formulaCH₃(CH₂)_(s)CO—NHCH((CH₂)₂COOH)CO—, wherein s is an integer of from 8 to24.
 111. The derivative of claim 94, wherein the lipophilic substituentis a group of the formula —NHCH(COOH)(CH₂)₄NH—CO(CH₂)_(u)CH₃, wherein uis an integer of from 8 to
 18. 112. The derivative of claim 94, whereinthe lipophilic substituent is a group of the formula—NHCH(COOH)(CH₂)₄NH—COCH((CH₂)₂COOH)NH—CO(CH₂)_(w)CH₃, wherein w is aninteger of from 10 to
 16. 113. The derivative of claim 94, wherein thelipophilic substituent is a group of the formula—NHCH(COOH)(CH₂)₄NH—CO(CH₂)₂CH(COOH)NH—CO(CH₂)_(x)CH₃, wherein x is aninteger of from 10 to
 16. 114. The derivative of claim 94, wherein thelipophilic substituent is a group of the formula—NHCH(COOH)(CH₂)₄NH—CO(CH₂)₂CH(COOH)NH—CO(CH₂)_(y)CH₃, wherein y is zeroor an integer of from 1 to
 22. 115. The derivative of claim 95, havingan amino acid sequence of HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGX, wherein X=Por Y, or a fragment or an analogue thereof.
 116. The derivative of claim95, having an amino acid sequence ofHX1X2GTFITSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS, wherein X1X2=SD or GE, or afragment or an analogue thereof.
 117. The derivative of claim 95, havingan amino acid sequence of DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS, or a fragmentor an analogue thereof.
 118. The derivative of claim 92, which is Arg¹⁸,Leu²⁰, Gln³⁴, Lys³³ (N^(ε)-(γ-aminobutyroyl(N^(α)-hexadecanoyl)))Exendin-4-(7-45)-NH₂.
 119. The derivative of claim 92, which is Arg³³,Leu²⁰, Gln³⁴, Lys¹⁸ (N^(ε)-(γ-aminobutyroyl(N^(α)-hexadecanoyl)))Exendin-4-(7-45)-NH₂.
 120. A pharmaceutical composition comprising aderivative of claim 92 and a pharmaceutically acceptable vehicle orcarrier.
 121. A method of treating insulin dependent or non-insulindependent diabetes mellitus in a patient in need of such a treatment,comprising administering to the patient a therapeutically effectiveamount of a derivative of claim 92 and a pharmaceutically acceptablecarrier.